scholarly journals Preclinical Evaluation of a Novel Orally Bioavailable Menin-MLL Interaction Inhibitor, DSP-5336, for the Treatment of Acute Leukemia Patients with MLL-Rearrangement or NPM1 Mutation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3339-3339
Author(s):  
Ken Eguchi ◽  
Takafumi Shimizu ◽  
Daiki Kato ◽  
Yudai Furuta ◽  
Seiji Kamioka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Vehicle Control ◽  
Human Leukemia ◽  
Npm1 Mutation ◽  
Significant Prolongation ◽  
Orally Bioavailable ◽  
Differentiation Marker

Abstract Background: The 11q23 abnormalities involving mixed lineage leukemia (MLL) gene are frequently found in adult and pediatric patients with acute leukemia. MLL rearrangements (MLL-r) are often associated with a poor prognosis and show poor response to currently available therapies, thus developing more effective therapy is urgently required. The leukemogenic activity of MLL fusion proteins, the products of the chimeric genes of MLL and its fusion partners generated by MLL-r, is critically dependent on direct interaction with MENIN, the product of the MEN1 gene. Interaction of MLL fusion proteins with MENIN plays an important role to enhance the proliferation and to block the differentiation of hematopoietic cells by maintaining high expression of hematopoietic stem cell program genes, such as HOXA9 and MEIS1. It has also been reported that the MENIN interaction with wild-type MLL, is required to induce HOXA9 and MEIS1 expression and also crucial for the development of acute leukemia with nucleophosmin (NPM1) mutations. Therefore, the MENIN-MLL interaction inhibitor is expected to have anti-leukemogenic activity against acute leukemia with MLL-r or NPM1 mutation by suppressing the expression of HOXA9 and MEIS1 and inducing terminal differentiation. Results: We generated DSP-5336, a novel, potent, and orally bioavailable MENIN-MLL interaction inhibitor for the treatment of acute leukemia patients with MLL-r or NPM1 mutation. DSP-5336 directly bound to the MENIN protein (Kd = 6.0 nM) and inhibited the MENIN-MLL interaction (IC 50 = 1.4 ± 0.058 nM). DSP-5336 selectively inhibited the cell growth of human leukemia cell lines including MV-4-11, MOLM-13, KOPN-8, and OCI-AML3 (IC 50 = 10, 15, 31 and 15 nM, respectively). These DSP-5336-sensitive cell lines possess a MLL-r or NPM1 mutation. On the other hand, DSP-5336 did not affect the cell growth of human leukemia cell lines such as HL-60, MOLT-4, and Reh (IC 50 > 10 μM), which do not have MLL-r or NPM1 mutations. In a mouse xenograft model using MV-4-11 cells, which express MLL-AF4, DSP-5336 exhibited a significant antitumor activity at the doses of 25 mg/kg and 50 mg/kg, administered twice daily (BID) for 20 days. There were no dose related changes in general condition or body weight. The effects of DSP-5336 on the expression of MENIN-MLL-regulated genes and differentiation marker genes were evaluated using MV-4-11 cells as pharmacodynamics markers in vitro and in vivo. In both cases, DSP-5336 significantly reduced the gene expression of MEIS1 and HOXA9, representative leukemic genes regulated by the MENIN-MLL complex. On the contrary, DSP-5336 significantly increased the gene expression level of ITGAM, a terminal differentiation marker. The efficacy of DSP-5336 was further assessed in acute leukemia patient samples and in mouse AML models. DSP-5336 strongly inhibited blast colony formation and changed the gene expression of the pharmacodynamics markers (HOXA9, MEIS1 and ITGAM ) in an AML patient sample carrying the MLL-AF6 fusion. In patient-derived xenograft (PDX) model with NPM1 mutation, human CD45 positive cells in peripheral blood progressively decreased during and beyond the 28 day period of DSP-5336 administration at doses of 25, 50, and 100 mg/kg BID and achieved a complete remission with no relapse at the doses of 50 and 100 mg/kg BID. At these three dose levels, DSP-5336 also induced a significant prolongation of survival compared to the vehicle control. Similarly, in a PDX model with MLL-AF4, DSP-5336 induced complete remission and significant prolongation of survival at the doses of 100 mg/kg BID compared to the vehicle control. In mouse AML models wherein MLL-ENL- or MLL-AF10-transduced bone marrow cells are transplanted in syngeneic mice, DSP-5336 induced a significant prolongation of survival at the doses of 200 mg/kg once daily (QD) compared to the vehicle control and the standard chemo therapy (cytarabine+daunorubicin) group. Summary: DSP-5336 has a potential as an antitumor drug that provides survival advantages in acute leukemia patients with MLL rearrangement or NPM1 mutation. Currently, a Phase 1/2 clinical study of DSP-5336 is planned in AML and ALL patients. Disclosures Eguchi: Sumitomo Dainippon Pharma: Current Employment. Shimizu: Sumitomo Dainippon Pharma: Current Employment. Kato: Sumitomo Dainippon Pharma: Current Employment. Furuta: Sumitomo Dainippon Pharma: Current Employment. Kamioka: Sumitomo Dainippon Pharma: Current Employment. Ban: Sumitomo Dainippon Pharma: Current Employment. Ymamoto: Sumitomo Dainippon Pharma: Current Employment. Yokoyama: Sumitomo Dainippon Pharma: Research Funding. Kitabayashi: Sumitomo Dainippon Pharma: Research Funding.

Effect of sodium dichloroacetate on apoptotic gene expression in human leukemia cell lines

2019 ◽  
Vol 71 (2) ◽  
pp. 248-256 ◽  
Author(s):  
Jagoda Abramek ◽  
Jacek Bogucki ◽  
Marta Ziaja-Sołtys ◽  
Andrzej Stępniewski ◽  
Anna Bogucka-Kocka
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Human Leukemia Cell ◽  
Apoptotic Gene ◽  
Leukemia Cell Lines

CBX8, a Polycomb-Group Protein, Is Essential for MLL-AF9-Induced Leukemogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4174-4174
Author(s):  
Jiaying Tan ◽  
Jay L. Hess
Keyword(s):  
Gene Expression ◽  
Cell Growth ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Polycomb Group ◽  
Human Leukemia ◽  
Interaction Sites

Abstract Abstract 4174 Trithorax and Polycomb-group (Trx-G and Pc-G) proteins are antagonistic regulators of homeobox-containing (Hox) gene expression that play a major role in regulation of hematopoiesis and leukemogenesis. Mixed lineage leukemia (MLL), a mammalian Trx-G protein, is a histone methyltransferase crucial for embryonic development and hematopoiesis that is commonly altered by translocation in acute leukemia. Recent evidence suggests that transformation by MLL fusion proteins is dependent on multiple interaction complexes, including the polymerase associated factor complex (PAFc) and the elongation activating protein complex (EAPc) or a closely related AF4 family/ENL family/P-TEFb complex (AEPc). CBX8 is a human PcG protein, functioning as a transcription repressor in the polycomb repressive complex 1 (PRC1). Previous studies have shown that CBX8 also interacts with the EAPc components AF9 and ENL; however, its role in leukemogenesis is unknown. To elucidate the significance of this interaction between these two proteins thought to have antagonistic function, we generated a large series of point mutations in AF9 and identified two amino acids that are essential for CBX8 interaction but preserve the interaction with other EAP components. Mutation of the two sites reduced the transcriptional activation of the MLL-AF9 target promoters by nearly 50% and completely inhibits the ability of MLL-AF9 to immortalize bone marrow (BM) as assessed by methylcellulose replating assays. This finding suggests that CBX8 interaction is essential for MLL-AF9-induced leukemogenesis. Several lines of evidence further support this finding. First, CBX8 knockdown by siRNAs decreased MLL-AF9-induced transcriptional activation by approximately 50%. Second, the ability of MLL-AF9 to transform primary BM was markedly reduced by retroviral shCbx8 transduction. Notably, this inhibitory effect is specific for MLL-AF9 because the BM transformation ability of E2A-HLF was unaffected by Cbx8 suppression. Third, Cbx8 suppression by shCbx8 in MLL-AF9 and MLL-ENL, but not E2A-HLF transformed AML cell lines, significantly inhibited the expression of MLL-dependent target genes, as well as cell growth and colony forming ability. Fourth, inducing CBX8 knockdown in human leukemia cell lines expressing MLL-AF9 led to a marked decrease in the localization of basic transcription machinery at the Hoxa9 locus and a corresponding reduction in Hoxa9 transcription. Importantly, the observed effects of CBX8 on MLL-rearranged leukemia cells are PRC1-independent: no effects on MLL target gene expression, cell growth, or BM transformation ability were observed by suppressing other core components of PRC1. Taken together, our results indicate that CBX8, independent of its transcription repression role in PRC1, interacts with and synergizes with MLL fusion proteins to promote leukemogenesis. Defining the interaction sites between AF9/ENL and CBX8 and the dependence of other AML subtypes and normal hematopoiesis on CBX8 will be important for the further development of agents that target this mechanism in MLL-rearranged and potentially other AML subtypes. Disclosures: No relevant conflicts of interest to declare.

Marked synergism of cytotoxic agents with either a prenylation inhibitor or M-TOR inhibitor in acute leukemia cell lines: Potential clinical implications

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 13103-13103
Author(s):  
D. R. Budman ◽  
A. Calabro
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Single Agent ◽  
Leukemia Cell ◽  
Cytotoxic Agents ◽  
Human Leukemia ◽  
Effect Analysis ◽  
Leukemia Cell Lines ◽  
Classical Combination

13103 Background: The most combinations of anticancer drugs are based upon empiricism. The potential permutations of drugs overwhelm the clinical trials system. Acute leukemia is sensitive to a variety of agents but relapses are common. Targeted agents are attractive new venues of therapy both as single agents and in combination with older agents. Isobologram median effect analysis allows up to three agents to be studied together in vitro to identify interesting combinations. We evaluated a commercially available statin, fluvastatin, to block prenylation which affects a variety of pathways, rapamycin and its experimental analogue RAD001 as M-TOR inhibitors to block downstream of the AKT pathway, and cytotoxic agents. Methods: The human leukemia cell lines AML-193 and KG-1 were obtained from ATTC (Rockville, MD), fluvastatin and RAD001 from Novartis Pharma, and the other agents from Sigma-Aldrich (St. Louis, MO). The IC50 of the single agent was determined by a 72 hr incubation of log growth cells using a MTT assay and the EZ-ED50 program (Perrella Scientific, Conyers, CA). The dosages of all agents were at clinically achievable concentrations. All reported values were the means of at least 3 experiments with each study using 4 wells per point. For isobologram analysis, a minimum of 8 concentrations of drug mixtures were studied above and below the IC50. Median effect CI values less than 1 are synergistic. Results: Doublets of fluvastatin with Ara-C (0.7), daunomycin (0.4), idarubicin (0.7), RAD001 (0.5), or rapamycin (0.3) demonstrated synergy. Doublets of RAD001 with Ara-C (0.3), daunomycin (0.7), or idarubicin (0.5) demonstrated synergy. Triplets of RAD001/daunorubicin/Ara-C, RAD001/daunomycin/fluvastatin, and RAD001/Ara-C/idarubicin all demonstrated marked synergy in both cell lines. Conclusion: A new potential non classical combination for further investigation is RAD001 or rapamycin with an inhibitor of prenylation such as fluvastatin. Additional potential combinations include cytotoxics with either fluvastatin or RAD001, and triplet combinations. No significant financial relationships to disclose.

scholarly journals Dynamin inhibition causes context-dependent cell death of leukemia and lymphoma cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0256708
Author(s):  
Christopher von Beek ◽  
Linnéa Alriksson ◽  
Josefine Palle ◽  
Ann-Marie Gustafson ◽  
Mirjana Grujic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Tumor Formation ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Bone Marrow Biopsies

Current chemotherapy for treatment of pediatric acute leukemia, although generally successful, is still a matter of concern due to treatment resistance, relapses and life-long side effects for a subset of patients. Inhibition of dynamin, a GTPase involved in clathrin-mediated endocytosis and regulation of the cell cycle, has been proposed as a potential anti-cancer regimen, but the effects of dynamin inhibition on leukemia cells has not been extensively addressed. Here we adopted single cell and whole-population analysis by flow cytometry and live imaging, to assess the effect of dynamin inhibition (Dynasore, Dyngo-4a, MitMAB) on pediatric acute leukemia cell lines (CCRF-CEM and THP-1), human bone marrow biopsies from patients diagnosed with acute lymphoblastic leukemia (ALL), as well as in a model of lymphoma (EL4)-induced tumor growth in mice. All inhibitors suppressed proliferation and induced pronounced caspase-dependent apoptotic cell death in CCRF-CEM and THP-1 cell lines. However, the inhibitors showed no effect on bone marrow biopsies, and did not prevent EL4-induced tumor formation in mice. We conclude that dynamin inhibition affects highly proliferating human leukemia cells. These findings form a basis for evaluation of the potential, and constraints, of employing dynamin inhibition in treatment strategies against leukemia and other malignancies.

Epigenetic modulation of gene expression of human leukemia cell lines – induction of cell death and senescence

Neoplasma ◽  
2011 ◽  
Vol 58 (1) ◽  
pp. 35-44 ◽  
Author(s):  
K. ELKNEROVA ◽  
D. MYSLIVCOVA ◽  
Z. LACINOVA ◽  
I. MARINOV ◽  
L. UHERKOVA ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Human Leukemia Cell ◽  
Leukemia Cell Lines ◽  
Modulation Of Gene Expression ◽  
Epigenetic Modulation

Upregulation of HLA-G Gene Expression in Malignant Hematopoietic Cells Treated with 5-Aza-2′- Ddeoxycytidine.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4567-4567
Author(s):  
Firas Alsabty ◽  
Martin Mistrik ◽  
Katarina Polakova
Keyword(s):  
Gene Expression ◽  
G Protein ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Dna Demethylation ◽  
Lymphocytic Leukemia ◽  
Human Leukemia ◽  
Rt Pcr ◽  
Demethylating Agent

Abstract Abstract 4567 Introduction Human leukocyte antigen G (HLA-G) is a nonclassic HLA class I antigen with restricted distribution in normal tissues. It exerts multiple immunregulatory functions that have been suggested to contribute to the immune evasion of tumour cells. Ectopic HLA-G expression observed in some pathological conditions such as malignant transformation may be triggered by epigenetic modifications such as DNA demethylation or histone acetylation. Materials and Methods Mononuclear cells were isolated from peripheral blood of newly diagnosed previously untreated patients with acute myeloblastic leukemia (AML) (n=9) and chronic lymphocytic leukemia (CLL) (n=5) by standard Ficoll-Hypaque density gradient centrifugation. Isolated cells were resuspended in RPMI 1640 medium supplemented with 2mM L-glutamine, 200 μg/ml gentamicin, 0.125 μg/ml amphotericin B and 10% heat-inactivated fetal bovine serum. Demethylating treatment of cells was carried out with100 μM 5-aza-2x- deoxycytidine (AdC) (Sigma) for 3 days. Human choriocarcinoma cell lines JEG3 and JAR (ATCC, Rockville, MD) were used as HLA-G positive and negative controls, respectively. Real time polymerase chain reaction (RT-PCR) and semiquantitative RT-PCR were performed using the ABI Prism 7000 Sequence Detection System and AmpliTaq Gold DNA polymerase to detect HLA-G mRNAs transcriptions. The HLA-G protein expression was examined by western blot analysis using mAb 4H84. Results HLA-G transcripts in AdC untreated leukemia samples were demonstrated in 3 out of 5 patients (60%) with B-CLL and in 5 out of 9 patients (56%) with AML. Treatment with demethylating agent AdC resulted in up-regulation of HLA-G transcription and expression of HLA-G protein in 5 out of 8 (63%) examined leukemia cell lines (Table 1). Conclusions we conclude that DNA methylation is an important control mechanism of HLA-G gene expression, and treatment of human leukemia with demethylating agent AdC may up-regulate HLA-G gene expression and induce HLA-G protein synthesis in some patients that may allow leukemic cells to escape recognition and destruction by cytotoxic T-cells or NK cells. Therefore patients should be monitored for HLA-G expression in order to follow risk of AdC therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Pre-Clinical Evaluation of the Proteasome Inhibitor Ixazomib against Bortezomib-Resistant Leukemia Cells and Primary Acute Leukemia Cells

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 665
Author(s):  
Margot S.F. Roeten ◽  
Johan van Meerloo ◽  
Zinia J. Kwidama ◽  
Giovanna ter Huizen ◽  
Wouter H. Segerink ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Unmet Need ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Cross Resistance ◽  
Proteasome Subunit

At present, 20–30% of children with acute leukemia still relapse from current chemotherapy protocols, underscoring the unmet need for new treatment options, such as proteasome inhibition. Ixazomib (IXA) is an orally available proteasome inhibitor, with an improved safety profile compared to Bortezomib (BTZ). The mechanism of action (proteasome subunit inhibition, apoptosis induction) and growth inhibitory potential of IXA vs. BTZ were tested in vitro in human (BTZ-resistant) leukemia cell lines. Ex vivo activity of IXA vs. BTZ was analyzed in 15 acute lymphoblastic leukemia (ALL) and 9 acute myeloid leukemia (AML) primary pediatric patient samples. BTZ demonstrated more potent inhibitory effects on constitutive β5 and immunoproteasome β5i proteasome subunit activity; however, IXA more potently inhibited β1i subunit than BTZ (70% vs. 29% at 2.5 nM). In ALL/AML cell lines, IXA conveyed 50% growth inhibition at low nanomolar concentrations, but was ~10-fold less potent than BTZ. BTZ-resistant cells (150–160 fold) displayed similar (100-fold) cross-resistance to IXA. Finally, IXA and BTZ exhibited anti-leukemic effects for primary ex vivo ALL and AML cells; mean LC50 (nM) for IXA: 24 ± 11 and 30 ± 8, respectively, and mean LC50 for BTZ: 4.5 ± 1 and 11 ± 4, respectively. IXA has overlapping mechanisms of action with BTZ and showed anti-leukemic activity in primary leukemic cells, encouraging further pre-clinical in vivo evaluation.

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