Mer Receptor Tyrosine Kinase Is Over-Expressed In and Contributes to Oncogenesis In Acute Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1390-1390
Author(s):  
Alisa B. Lee-Sherick ◽  
Kristen M. Eisenman ◽  
Susan Sather ◽  
Deborah DeRyckere ◽  
Jennifer Schlegel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Targeted Therapy ◽  
Tyrosine Kinase ◽  
Western Blot ◽  
Cell Survival ◽  
Cell Lines ◽  
Wild Type ◽  
Knock Down ◽  
Acute Myelogenous

Abstract Abstract 1390 The abnormal activation of tyrosine kinases in pediatric leukemias has been associated with a poor prognosis, and provides a potential focus for targeted therapy. Pediatric acute myelogenous leukemia (AML) is known to be particularly difficult to treat successfully. The development of therapy for AML targeted against a specific cancer-promoting signaling pathway would potentially allow for a more efficacious clinical response with less therapy-associated toxicity. The Mer Tyrosine Kinase (TK), a transmembrane receptor in the TAM family, is known to regulate intracellular pathways promoting cell survival and proliferation in a number of malignancies, but has not previously been explored in AML. We assessed the prevalence of Mer TK expression in AML. Western blot and flow cytometric analysis demonstrated aberrant expression of Mer TK in 80% (13 of 15) of AML cell lines. Similarly, greater than 85% (24 of 28) of samples from newly diagnosed pediatric AML patients expressed Mer TK on leukemic blasts. In addition, 5 of 6 pediatric patients with relapsed or refractory AML had increased or equivalent Mer expression by flow cytometry relative to diagnostic samples. To assess whether Mer plays a role in proliferation in AML, we investigated downstream signaling pathways in the Nomo-1 and Kasumi-1 AML cell lines. Phosphoarray and western blot analysis demonstrated increased phospho-Erk 1/2, phospho-Akt, phospho-mTOR and phospho-MSK1 following treatment with Gas6, the Mer ligand. These data demonstrate activation of pathways which are known to aid in malignant cell survival. To assess the effect of Mer TK inhibition on myeloblast phenotype, we used two different shRNA constructs to decrease expression of Mer by >50% in the Nomo-1 and Kasumi-1 cell lines. The ability of these cell lines to evade apoptosis was determined by flow cytometry following staining with propidium iodide and Yo-Pro-1-iodide. Compared to wild-type Nomo-1 and Kasumi-1, the cell lines expressing decreased levels of Mer demonstrated two to four times more apoptosis in response to serum starvation (p<0.5). Additionally, myeloblast proliferative capacity was assessed using methylcellulose colony forming assays. Compared to wild-type, the AML cell lines expressing reduced levels of Mer demonstrated a 40–70% decrease in total colony forming units (p<0.5). To explore how knockdown of Mer affects myeloblast survival in vivo, we used a mouse xenograft model. Sub-lethally irradiated NSG mice were injected intravenously with wild-type Nomo-1 or Mer knock-down Nomo-1 lines and tumor-free survival was determined. Kaplan-Meier curves were generated and demonstrated a statistically significant difference in survival between mice injected with wild-type Nomo-1 cells and those injected with a Nomo-1 Mer knock-down cell line (20 versus 43 days, p<0.1). These data demonstrate a role for Mer in acute myelogenous leukemogenesis in vivo and suggest that inhibition of Mer TK may have a clinically significant effect in patients as a targeted therapy in the treatment of human AML. Disclosures: No relevant conflicts of interest to declare.

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.

CHIR258, a Novel Multi-Targeted Tyrosine Kinase Inhibitor, for the Treatment of t(4;14) Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 641-641 ◽  
Author(s):  
Suzanne Trudel ◽  
Zhi Hua Li ◽  
Ellen Wei ◽  
Marion Wiesmann ◽  
Katherine Rendahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Myeloma Cells

Abstract The t(4;14) translocation that occurs uniquely in a subset (15%) of multiple myeloma (MM) patients results in the ectopic expression of the receptor tyrosine kinase, Fibroblast Growth Factor Receptor3 (FGFR3). Wild-type FGFR3 induces proliferative signals in myeloma cells and appears to be weakly transforming in a hematopoeitic mouse model. The subsequent acquisition of FGFR3 activating mutations in some MM is associated with disease progression and is strongly transforming in several experimental models. The clinical impact of t(4;14) translocations has been demonstrated in several retrospective studies each reporting a marked reduction in overall survival. We have previously shown that inhibition of activated FGFR3 causes morphologic differentiation followed by apoptosis of FGFR3 expressing MM cell lines, validating activated FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these poor-prognosis patients. CHIR258 is a small molecule kinase inhibitor that targets Class III–V RTKs and inhibits FGFR3 with an IC50 of 5 nM in an in vitro kinase assay. Potent anti-tumor and anti-angiogenic activity has been demonstrated in vitro and in vivo. We employed the IL-6 dependent cell line, B9 that has been engineered to express wild-type FGFR3 or active mutants of FGFR3 (Y373C, K650E, G384D and 807C), to screen CHIR258 for activity against FGFR3. CHIR258 differentially inhibited FGF-mediated growth of B9 expressing wild-type and mutant receptors found in MM, with an IC50 of 25 nM and 80 nM respectively as determined by MTT proliferation assay. Growth of these cells could be rescued by IL-6 demonstrating selectivity of CHIR258 for FGFR3. We then confirmed the activity of CHIR258 against FGFR3 expressing myeloma cells. CHIR258 inhibited the viability of FGFR3 expressing KMS11 (Y373C), KMS18 (G384D) and OPM-2 (K650E) cell lines with an IC50 of 100 nM, 250 nM and 80 nM, respectively. Importantly, inhibition with CHIR258 was still observed in the presence of IL-6, a potent growth factors for MM cells. U266 cells, which lack FGFR3 expression, displayed minimal growth inhibition demonstrating that at effective concentrations, CHIR258 exhibits minimal nonspecific cytotoxicity on MM cells. Further characterization of this finding demonstrated that inhibition of cell growth corresponded to G0/G1 cell cycle arrest and dose-dependent inhibition of downstream ERK phosphorylation. In responsive cell lines, CHIR258 induced apoptosis via caspase 3. In vitro combination analysis of CHIR258 and dexamethasone applied simultaneously to KMS11 cells indicated a synergistic interaction. In vivo studies demonstrated that CHIR258 induced tumor regression and inhibited growth of FGFR3 tumors in a plasmacytoma xenograft mouse model. Finally, CHIR258 produced cytotoxic responses in 4/5 primary myeloma samples derived from patients harboring a t(4;14) translocation. These data indicate that the small molecule inhibitor, CHIR258 potently inhibits FGFR3 and has activity against human MM cells setting the stage for a Phase I clinical trial of this compound in t(4;14) myeloma.

A Small Molecule Stat Inhibitor Blocks Stat3 and Stat5 Phosphorylation and Demonstrates Cytotoxicity In Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2904-2904
Author(s):  
Robyn M. Dennis ◽  
Brandon Ballard ◽  
David John Tweardy ◽  
Karen Rabin
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Flow Cytometric Analysis ◽  
Positive Control ◽  
Wild Type ◽  
Atp Assay ◽  
Antibody Staining

Abstract Abstract 2904 Survival has improved dramatically in acute lymphoblastic leukemia (ALL), but further gains are unlikely using conventional chemotherapy alone. Several recently discovered, novel cytogenetic lesions with adverse prognostic impact, JAK2 activating mutations and CRLF2 rearrangements, occur in up to 15% of adult and pediatric ALL. These lesions are associated with activation of Jak2 and Stat5, and hold promise as targets for novel therapies affecting these signaling pathways. We performed in vitro testing of a novel small molecule Stat inhibitor, C188-9, in B-lineage ALL cell lines and patient samples with and without JAK2/CRLF2 alterations. C188-9 treatment for one hour at 10 μM inhibited Stat3 and Stat5 phosphorylation in ALL cell lines with JAK2 and CRLF2 alterations, but not in cell lines with wild-type JAK2 and CRLF2, as measured by phospho-flow cytometry (Fig. 1A). Only the cell lines with JAK2 and CRLF2 alterations demonstrated basal Stat5 phosphorylation on Western blot analysis, and this was inhibited by C188-9 treatment (Fig. 1B). C188-9 demonstrated cytotoxicity in ALL cell lines regardless of JAK2/CRLF2 status, with IC50s in the low micromolar concentration range (Fig. 1C). While C188-9 is undergoing investigation currently as a potent inhibitor of Stat3 in acute myeloid leukemia (AML), it also merits further investigation as an agent with Stat5 inhibitory activity and cytotoxicity in ALL. Figure 1. Effects of C188-9 in ALL cell lines. A. Stat3 and Stat5 phosphorylation were determined by flow cytometry in the ALL cell lines MHH-CALL-4 (JAK2/CRLF2 mutated) and Reh (JAK2/CRLF2 wild-type). In each condition, cells were incubated in serum-free media for one hour, followed by incubation with C188-9 or vehicle for one hour, stimulation with vehicle or pervanadate 125 mM for 15 minutes, fixation, permeabilization, phospho-antibody staining for phospho-Stat3 and phospho-Stat5, and flow cytometric analysis. B. Western blot for phospho-Stat5 in K562 cell line (positive control); MHHCALL-4 treated for one hour with C188-9 at 0, 5, or 10 uM; and RS4;11 (JAK2/CRLF2 wild-type ALL cell line). C. IC50 determination by ATP assay for C188-9 in the ALL cell lines MHH-CALL-4 and RS4;11. Each experiment was performed in triplicate. Figure 1. Effects of C188-9 in ALL cell lines. A. Stat3 and Stat5 phosphorylation were determined by flow cytometry in the ALL cell lines MHH-CALL-4 (JAK2/CRLF2 mutated) and Reh (JAK2/CRLF2 wild-type). In each condition, cells were incubated in serum-free media for one hour, followed by incubation with C188-9 or vehicle for one hour, stimulation with vehicle or pervanadate 125 mM for 15 minutes, fixation, permeabilization, phospho-antibody staining for phospho-Stat3 and phospho-Stat5, and flow cytometric analysis. B. Western blot for phospho-Stat5 in K562 cell line (positive control); MHHCALL-4 treated for one hour with C188-9 at 0, 5, or 10 uM; and RS4;11 (JAK2/CRLF2 wild-type ALL cell line). C. IC50 determination by ATP assay for C188-9 in the ALL cell lines MHH-CALL-4 and RS4;11. Each experiment was performed in triplicate. Disclosures: No relevant conflicts of interest to declare.

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.

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.

The Tyrosine Kinase Src Regulates Adhesion and Survival in Waldenström Macroglobulinemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2615-2615
Author(s):  
Hai T. Ngo ◽  
Mena Farag ◽  
Xiaoying Jia ◽  
Nicolas Burwick ◽  
Molly R. Melhem ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Src Kinase ◽  
Significant Inhibition ◽  
Thymidine Uptake ◽  
Inhibition Of Proliferation ◽  
Src Inhibitor

Abstract BACKGROUND: Tyrosine phosphorylation is a central event in the regulation of a variety of biological processes such as cell proliferation, migration, adhesion, and survival. Waldenström Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy and characterized by widespread involvement of the bone marrow. We sought to determine whether the protein tyrosine kinase Src regulates adhesion and survival in WM. METHODS: The WM cell lines BCWM1, WM-WSU, and IgM secreting lymphoma cells lines MEC-1 and RL were used in these studies. Primary CD19+ WM cells were obtained from the bone marrow of patients after informed consent. AstraZeneca Biopharmaceuticals (London, England) provided the Src inhibitor AZD0503 (AZD). Cytotoxicity and DNA synthesis were measured using MTT assay and [3H]-thymidine uptake, respectively. Apoptosis was measured using flow cytometry with Apo 2.7 staining. Western blotting was performed to determine downstream signaling pathways. Migration was performed using the transwell migration assay. RESULTS: We demonstrated that pSrc is overexpressed in WM cells compared to control B cells. Similarly, phospho-Src protein expression was upregulated in WM cell lines, specifically BCWM.1 but not in WM-WSU. We then showed that pSrc regulates migration and adhesion in response to the chemokine SDF-1, as well as in vivo homing using in vivo flow cytometry. The use of the specific Src inhibitor AZD0530 led to significant inhibition of adhesion and migration in cell lines with pSrc activation, but not in those deficient of Src activation. Similarly, inhibition of Src activity led to significant inhibition of proliferation and survival through inhibition of STAT3, Akt, and ERK/MAPK pathways. The monoclonal antibody rituximab signals through Src kinase, and the combination of AZD0530 and rituximab was synergistic in vitro. CONCLUSION: Taken together, these studies delineate the role of Src kinase activity in WM and provide the framework for future clinical trials using Src inhibitors in combination with rituximab to improve the outcome of patients with WM.

MLN4924, an Investigational Small Molecule Inhibitor of NEDD8-Activating Enzyme (NAE), Inhibits NF-κB Activity and Induces G1 Cell Cycle Arrest and Apoptosis in Pre-Clinical Models of Hodgkin Lymphoma (HL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3717-3717
Author(s):  
Matthew J. Barth ◽  
Cory Mavis ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Myron S. Czuczman
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Western Blot ◽  
Cell Viability ◽  
Tumor Cells ◽  
Cell Lines ◽  
Small Molecule ◽  
Wild Type ◽  
Cycle Arrest ◽  
Molecule Inhibitor

Abstract Abstract 3717 The incorporation of combined-modality therapy, risk-stratified chemotherapy selection, high-dose chemotherapy and autologous stem cell support (HDC-ASCS), and monitoring treatment response by functional imaging are factors that have contributed to the improvement in clinical outcomes in HL patients. Unfortunately, those patients not eligible for or that have failed HDC-ASCS remain a challenge for the treating oncologist, stressing the need for novel therapeutic strategies. Significant improvements in the understanding of the biology of HL have been achieved, including cellular pathways altered in HL (e.g. the ubiquitin-proteasome system) and the role of the tumor microenvironment. MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE). NAE is an enzyme responsible for activating NEDD8, an ubiquitin-like molecule in the neddylation cascade that is responsible for cullin-ring ligase (CRL) mediated polyubiquitination of proteins targeting them for proteasomal degradation. In order to better understand the activity of MLN4924 in HL, we performed pre-clinical testing in IkB wild type (L-1236), IkB mutated (KM-H2 and L-428) HL cell lines, and in primary tumor cells derived from a HL patient. Malignant cells were exposed to escalating doses of MLN4924 and changes in cell viability were quantified at different time periods by alamar Blue reduction assay. Patient tumor cells were incubated with MLN4924 for 48 hrs and cell viability was determined using the CellTiterGlo assay. Induction of apoptosis in HL cell lines following exposure to MLN4924 was determined by flow cytometry for Annexin-V and propidium iodide (PI) staining and western blot for caspase-3 and PARP cleavage. Cell cycle analysis was performed by flow cytometry using PI staining. Inhibition of NAE by MLN4924 in HL cell lines was measured by western blot for NEDD8-cullin. Finally, changes in NF-kB activity following MLN4924 exposure were determined by p65 nuclear localization using Image stream technology. MLN4924 exhibited a dose- and time-dependent decrease in cell viability in all HL cell lines at nM concentrations. No differences in anti-tumor activity were observed between IkB-wild type (L-1236 IC50 = 250nM) and IkB–mutated HL cell lines (KM-H2 IC50 = 250nM and L-428 IC50 = 300nM). MLN4924 induced apoptosis in a dose-dependent manner in all cell lines tested. In addition, MLN4924 induced cell cycle arrest in G1 phase and inhibition of NAE was demonstrated by a decrease in NEDD8 conjugated CRL. L1236 cells exposed to MLN4924 also demonstrated a decrease in degradation of IκBα as evidenced by increased levels of p-IκBα following exposure to MLN4924 with a corresponding decrease in p65 nuclear translocation. Surprisingly KMH-2 cells, which carry a mutated IκBα protein that is truncated and non-functional, had a decrease in nuclear p65 following exposure to MLN4924, suggesting an alternative mechanism of NF-kB inhibitory activity by MLN4924. In summary, MLN4924 demonstrates activity against HL cells in vitro through inhibition of NF-kB, and is a promising novel agent for the treatment of HL. We continue to investigate the pre-clinical activity of MLN4924 both as a single-agent and in combination with traditional chemotherapy and other novel agents. Disclosures: No relevant conflicts of interest to declare.

scholarly journals TP-0184, a Novel FLT3-ALK2 Dual Inhibitor, Targets Amino Acid Transport and Biosynthesis in AML Cells and Sensitizes AML Cells to Chemotherapy and BCL2 Inhibition

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 376-376
Author(s):  
Anudishi Tyagi ◽  
Stanley Ly ◽  
Bin Yuan ◽  
Fouad El-Dana ◽  
Appalaraju Jaggupilli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Targeted Therapy ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Wild Type ◽  
Dual Inhibitor ◽  
Serine Biosynthesis

Abstract Background FMS-like tyrosine kinase 3 (FLT3), a transmembrane receptor tyrosine kinase that is frequently mutated in AML, is associated with poor prognosis. Inhibitors of FLT3 internal tandem duplication (ITD) mutants and wild-type (WT) FLT3 have been studied, but their clinical usefulness is limited owing to treatment resistance. However, the molecular factors contributing to this resistance are unknown. We reported that AML cells induce osteogenic differentiation of bone marrow-derived mesenchymal stromal cells through the bone morphogenetic protein (BMP)-mediated signaling pathway, promoting leukemic growth. However, the effects of targeting BMP signaling in patients with FLT3-mutated AML are unexplored. Here, we hypothesized that the BMP type 1 receptor ALK2 is a key biomarker and a therapeutic target in AML patients with FLT3-ITD mutations and that the FLT3-ALK2 dual inhibitor TP-0184 inhibits leukemia growth. Methods: To determine whether ALK2 is a potential target in AML patients with FLT3-ITD mutations, we analyzed gene expression datasets (OHSU and TCGA). We treated 9 AML cell lines with FLT3-WT or ITD mutations with varying doses of ALK2 inhibitors (LDN-212854 or TP-0184) and measured their effect on cell proliferation and the cell cycle. To determine the mechanism of TP-0184-mediated cell cycle arrest, we measured activation of FLT3 downstream signaling by using Western blotting and RNA sequencing. IncuCyte live-cell imaging was used to determine the apoptotic effects of TP-0184 in combination with chemotherapy or targeted therapy agents. Further, we performed human RTK kinase binding assay to understand the binding specificity of TP-0184 with 11 different FLT3 mutants. Finally, the effect of TP-0184 on AML growth in vivo was investigated using an FLT3-ITD positive AML xenograft model (MOLM13). Results: Analysis of AML datasets showed that ALK2 is significantly upregulated in AML patients with FLT3 mutations compared to those with WT-FLT3 (p &lt; 0.00001) and predicts poor overall survival (p = 0.05). Validating these findings, we found higher ALK2 mRNA expression in AML cell lines with FLT3-ITD mutations than in those with WT-FLT3 (p = 0.039). This suggests that ALK2 could serve as a therapeutic target in AML with FLT3-ITD mutation. Treatment of FLT3-WT and -mutated AML cell lines with the ALK2 inhibitors LDN-212854 and TP-0184 resulted in significant inhibition of FLT3-ITD-mutant cell growth at low concentrations (IC50&lt;25nM), while WT-FLT3 cells were affected only at high concentrations (IC50&gt;100nM). Interestingly, TP-0184 was 10-fold more potent in inhibiting AML cell proliferation than was LDN-212854. TP-0184 induced G1/G0 arrest in AML cell lines with FLT-ITD mutations but had minimal to no effect in FLT3-WT AML cells, suggesting that AML cell lines with FLT3-ITD mutations depend on ALK2 for their survival. Further, we observed that treatment with TP-0184 in AML cell lines significantly inhibited multiple signaling proteins downstream of FLT3, such as p-STAT5, p-MKK3, and p-ERK, as well as p-PI3K, p-AKT, p-mTOR, p-4E-BP1, and p-S6K. Gene expression analysis revealed that treatment with TP-0184 in FLT3-ITD cell lines significantly downregulated the serine biosynthesis pathway, which is essential in these cells (Bjelosevic S. et al., Cancer Discov, 2021). Moreover, molecular docking and kinase-binding studies revealed that TP-0184 is bound to wild-type FLT3 as well as most of the FLT3 mutants with dissociation constants (KD) less than 5nM. These data suggest that TP-0184 inhibits both mutant FLT3 and ALK2 in AML cells. Interestingly, TP-0184 plus chemotherapy showed a synergistic effect only in FLT3-ITD cell lines, whereas TP-0184 plus the BCL2 inhibitor, venetoclax was synergistic in both FLT3-ITD and FLT3-WT cell lines. Lastly, treatment with TP-0184 inhibited AML growth and significantly prolonged survival of FLT3-ITD-mutated AML-bearing mice in a dose-dependent manner (p &lt;0.0001). Conclusion: Our data indicate that ALK2 is a prognostic marker for AML patients with FLT3-ITD mutations. TP-0184 significantly inhibits cell proliferation by inhibiting signaling pathways downstream of FLT3, including serine biosynthesis, in AML cells. Kinase assays confirmed that TP-0184 is a highly specific FLT3 inhibitor as well as an ALK2 inhibitor. TP-0184 sensitizes AML cells to chemotherapeutic agents and targeted therapy and inhibits AML growth in vivo. Disclosures Foulks: Sumitomo Dainippon Pharma Oncology: Patents & Royalties: WO2021102343A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: CA3103995A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: US11040038B2. Warner: Sumitomo Dainippon Pharma Oncology: Patents & Royalties: US11040038B2; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: US10752594B2; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: CA3103995A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: WO2021102343A1. Battula: Tolero Pharmaceuticals: Research Funding.

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.

Activity of the Multi-Targeted Kinase Inhibitor, AT9283 on Imatinib-Resistant CML Models.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1104-1104 ◽  
Author(s):  
Ruriko Tanaka ◽  
Matthew S Squires ◽  
Shinya Kimura ◽  
Asumi Yokota ◽  
Kirsty Mallett ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Wild Type ◽  
Once Daily ◽  
Imatinib Resistant ◽  
Mutant Forms ◽  
In Vivo Effects

Abstract CML is caused by a consistent genetic abnormality, termed the Philadelphia chromosome, that results from a reciprocal (9;22) translocation leading to the expression of the BCR-ABL fusion protein. Although treatment has been revolutionized by the introduction of tyrosine kinase inhibitors which target Abl activity, reactivation of Abl signaling via several different point mutations remains problematic. In particular the mutation of Threonine 315 to Isoleucine (T315I) confers resistance to all existing therapies with tyrosine kinase inhibitors in the clinical settings. We describe the in vitro and in vivo effects of AT9283, a potent inhibitor of several protein kinases, including Abl kinase (wild type BCR-ABL and several of the drug resistant mutant variants that have arisen in clinical practice e.g. T315I), JAK2, JAK3 and Aurora kinases A and B, on imatinib-resistant CML cells including those harboring BCR-ABL (T315I). AT9283 has potent anti-proliferative activity in a panel of BaF3 and human cell lines expressing the BCR-ABL or its mutant forms. In BaF3 BCR-ABL wild-type and T315I mutant cells and K562 CML cells we observed inhibition of substrates of both BCR-ABL (STAT5) and Aurora B (Histone H3) at concentrations &gt;300nM and &lt;100nM, respectively, suggesting that AT9283 is capable of inhibiting Aurora and BCR-ABL simultaneously in these cell lines. The in vivo effects of AT9283 were examined in several mouse models engrafted either subcutaneously or intravenously with BaF3, human CML cell lines or primary CML patient samples expressing the BCR-ABL or its mutant forms. Specifically AT9283 prolonged the survival of mice engrafted intravenously with either BaF3 BCR-ABL T315I, or E255K cells when administered intraperitoneally twice daily at doses of either 6.25 or 10mg/kg or once daily at 15mg/kg when administered 5 days in every week repeated twice. Maximal survival advantage was conferred at either 10mg/kg twice daily or 15mg/kg once a day. Similar data were obtained in an intravenous model using primary CML cells taken from a patient harbouring the BCR-ABL E255K mutation. We also present data from ongoing studies showing increased survival rates in these in vivo model systems following multiple cycles of AT9283 administered on the 15mg/kg once daily schedule. These data together support further clinical investigation of AT9283 in patients with treatment resistant CML.

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