MI-63: A novel small-molecule inhibitor targets MDM2 and induces apoptosis in embryonal and alveolar rhabdomyosarcoma cells with wild-type p53

Abstract Abstract 386 Heterozygous mutations in the neutrophil elastase gene ELANE have been identified as the primary cause of severe congenital neutropenia (SCN) associated with recurring severe infections and evolution to acute myeloid leukemia (AML). As of today, more than 50 substitution, truncation, insertion and deletion mutations have been identified. Animal studies based on knock-in or knockout of ELANE in mice failed to produce severe neutropenia phenotype. We and others previously reported that expression of various mutants but not wild type neutrophil elastase (NE) in human but not murine cells triggers accelerated apoptosis. We also reported that expression of mutant NE (del.145-152), identified in SCN patients one of whom evolved to develop MDS/AML, in human promyelocytic tet-off HL60 cells causes both accelerated apoptosis and characteristic block of myeloid differentiation similar to that seen in bone marrow of SCN patients. Examination of the tertiary structure of NE revealed that most of the mutations leave the active site of the mutant protease intact. We identified a small molecule inhibitor of neutrophil elastase, a derivative of L-malic acid (Merck, USA), that blocked the proteolytic activity of NE by approximately 80% and was capable of restoring impaired myeloid differentiation and normalizing production of myeloid cells expressing del145-152 NE mutant. It is important to note that block of proteolytic activity of NE with the NE-SMI had no adverse effect on control human myeloid progenitor cells expressing wild type NE, thus confirming the gain-of-function effect of NE mutants. More than 20% of SCN patients with NE mutations evolve to develop AML. Molecular modeling and analysis of the tertiary structures of NE available through the Protein Database revealed that 16 different mutations identified in AML patients affect predominantly the N95 or N144 glycosylation sites or the binding pocket of the protease suggesting that altered substrate specificity of the mutant enzyme is the cause of accelerated apoptosis and block of myeloid differentiation in SCN/AML. We sought to obtain bone marrow samples from 2 unrelated SCN/AML patients both on G-CSF treatment harboring either C122Y or insPQ94. Bone marrow purified CD34+ and/or CD34-/CD33+ myeloid progenitors from the patients showed basal level of apoptosis in a range of 20–25%, which gradually increased reaching 40–50% apoptosis by 3 days of culture. Importantly, treatment of primary bone marrow-derived cells with NE-SMI substantially reduced accelerated apoptosis to near initial rate with approximately up to 2-fold reduction of apoptosis by 3 days of culture as determined by flow cytometry. Thus, our findings demonstrate that 1) small molecule inhibitor of neutrophil elastase is effective in blocking accelerated apoptosis triggered by three different NE mutations identified in SCN patients evolved to develop MDS/AML and 2) the small molecule inhibitor of NE is a promising therapeutic agent that should be considered for testing in clinical trials in SCN/AML patients. Disclosures: Dale: Amgen: Consultancy, Research Funding; Merck: Patents & Royalties, Research Support.

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Small-Molecule Inhibitor Screen Rapidly Identifies Key Signaling Pathways in Leukemogenesis

Blood ◽

10.1182/blood.v112.11.2519.2519 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2519-2519

Author(s):

Jeffrey W Tyner ◽

Heidi Erickson ◽

Stephen Oh ◽

Jason R. Gotlib ◽

Michael W.N. Deininger ◽

...

Keyword(s):

Tyrosine Kinase ◽

Cell Viability ◽

Small Molecule ◽

Tyrosine Kinases ◽

Small Molecule Inhibitor ◽

Wild Type Allele ◽

Wild Type ◽

Kinase Activation ◽

Type Allele ◽

Molecule Inhibitor

Abstract Aberrantly activated tyrosine kinases and their associated signaling pathways are critical to leukemogenesis and primary acute myeloid leukemia (AML) cell viability. While aberrant kinase activation has been confirmed in a significant percentage of AML, constitutive phosphorylation of STAT5, a marker of tyrosine kinase activation, is present in the majority of AML samples indicating that as yet unidentified tyrosine kinases can be aberrantly activated and contribute to leukemogenesis. Efforts to identify activating tyrosine kinase mutations using high-throughput sequencing have identified low frequency mutations of uncertain functional significance. Because these studies failed to detect additional high-frequency kinase mutations, the identity and mechanism of tyrosine kinase activation may be unique in many AMLs. Methods: To rapidly identify activated kinase pathways in individual, primary AML samples, we have developed a small-molecule inhibitor array which includes 90 small-molecule, cell-permeable inhibitor compounds including a core of 36 tyrosine kinase inhibitors that covers the majority of the tyrosine kinome. Many of the inhibitors are available for clinical use or are in clinical development. In this assay, inhibitors were placed in 96-well plates at four serial dilutions to allow IC50 calculations. Three days after adding primary AML cells to each well, we performed an MTS cell viability assay to evaluate the effect of each inhibitor on cell viability. Because most inhibitors affect multiple kinases, we compared target specificities of compounds that decrease primary AML cell viability with those that have no effect to identify potential targets. Results: In preliminary proof-of-principal experiments, we tested leukemic cells from five AML patients, including three that were positive for FLT3 internal tandem duplication (ITD), a genetic lesion that is thought to confer a proliferative advantage in approximately 30% of AML patients. Only one sample showed a clear response to small-molecule inhibitors known to target FLT3. The IC50s for the known FLT3 inhibitors MLN518, AST487, CHIR258, Sunitinib, and SU14813 were 10 to 100 fold lower in the leukemic cells from this patient than for the mean and median values for bone marrow samples from normal marrow samples. Interestingly, neither of the remaining FLT3-ITD positive leukemias nor the FLT3 negative leukemias demonstrated decreased viability in the presence of small-molecule inhibitors that target FLT3. A PCR based screen to identify FLT3-ITD alleles showed a near absence of the wild-type FLT3 allele in this sample, while the wild-type allele was present at equal or greater intensity as the ITD allele in the remaining FLT3-ITD positive AML samples. FLT3-ITD positive leukemias with loss of the FLT3 wild-type allele have been shown to have a poorer prognosis than those retaining a wild-type allele indicating aberrantly activated FLT3 may play a crucial role in leukemic cell viability in this setting and are consistent with a previous report showing that efficacy of the FLT3 inhibitor CEP-701 was greatest in pediatric AML with high FLT3-ITD mutant-to-wild-type allelic ratios. Though the remaining AML samples did not show a pattern of inhibitor sensitivity consistent with FLT3 activation, these and many of approximately 15 additional analyzed AML samples showed unique sensitivity patterns implicating other specific kinase targets or kinase families for further investigation while simultaneously providing therapeutic options. Conclusions: These preliminary data demonstrate that the small-molecule inhibitor functional assays can rapidly identify disease causing genes, provide insights into their mechanism of action, and suggest therapeutic options. The distinct patterns of tyrosine kinase sensitivity in these samples support the hypothesis that tyrosine kinases and related pathways contributing to leukemogenesis in each patient may be different and that targeted therapy will be most effective when administered on an individualized basis.

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Mi-63, a Small Molecule Inhibitor of MDM2-p53 Interaction, Has Significant In Vitro Activity in Multiple Myeloma.

Blood ◽

10.1182/blood.v108.11.3464.3464 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3464-3464

Author(s):

Shaji Kumar ◽

Michael Timm ◽

Michael P. Kline ◽

Jessica L. Haug ◽

Teresa K. Kimlinger ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Cell Lines ◽

Small Molecule ◽

Myeloma Cell ◽

Small Molecule Inhibitor ◽

Wild Type ◽

Myeloma Cells ◽

Molecule Inhibitor

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. The tumor suppressor gene p53, responsible for induction of cellular apoptosis in response to genotoxic stimuli, is relatively intact in most cases of myeloma. However, p53 mutations or deletion can occur late in the course of disease. Here we evaluate a novel small molecule inhibitor of the interaction between p53 and its negative regulator, MDM2, in the setting of myeloma. Methods and Results: Mi-63 was cytotoxic to several different myeloma cell lines with a median effect observed at approximately 2.5 μM in cell lines including MM1.S that express wild type p53 and between 10–15 μM in cells with mutated p53 as measured using an MTT cell viability assay. Additionally, Mi63 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. Mi63 was cytotoxic to myeloma cells under these conditions as well, at doses similar to those seen with myeloma cells alone. Mi63 was able to inhibit proliferation and induce apoptosis in myeloma cells in a dose- and time-dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. Treatment of myeloma cells with Mi63 was associated with early mitochondrial membrane depolarization, inversion of Bax/Bcl-2 ratio, and down regulation of Mcl-1, indicating induction of mitochondrial mechanisms of cell death. Mi63 was also cytotoxic to freshly isolated primary patient myeloma cells, inducing apoptosis in a dose-dependent manner. In the patient cells the drug appears to have a differential effect on the CD45 positive and negative cells. Conclusion: Mi-63 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. It clearly induces apoptosis in myeloma cells, with higher activity seen in cells with wild type p53. Given the lack of p53 abnormalities in most of the patients with myeloma, this drug alone or in combination is likely to have significant clinical activity. Studies combining this with various DNA damaging drugs are in progress. These studies will eventually form the framework for future clinical studies.

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Preclinical Evaluation of AT219, a Small Molecule Inhibitor of MDM2 As an Anti-Myeloma Agent.

Blood ◽

10.1182/blood.v120.21.2948.2948 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2948-2948

Author(s):

Vijay G. Ramakrishnan ◽

Teresa K. Kimlinger ◽

Timothy Halling ◽

Jessica Haug ◽

Utkarsh Painuly ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Small Molecule ◽

Research Funding ◽

Primary Cells ◽

Wild Type ◽

Drug Induced ◽

Molecule Inhibitor ◽

Wild Type P53 ◽

Stock Solutions

Abstract Abstract 2948 Background: Deletions and mutations in the tumor suppressor protein p53 are an uncommon observation in new multiple myeloma (MM) patients and are observed more commonly in patients with advanced disease. p53 deletion has been observed to correlate with poor overall and progression free survival in MM patients. Wild type p53 modulates the expression levels of a broad array of proteins involved in cell cycle progression, apoptosis ultimately leading to cell cycle arrest and apoptosis. p53 is negatively regulated by MDM2. MDM2 binds to and ubiquitinates p53 marking it for proteasomal degradation. In addition, MDM2 is a direct downstream regulator of p53. Targeting the p53-MDM2 interaction by developing agents that bind to the p53 binding motif of MDM2 and reactivating p53 has therefore been an active area of research. Here, we present results from our pre-clinical studies using AT219, a small molecule inhibitor that binds to MDM2 preventing its interaction with p53. Methods: AT219 was obtained from Ascenta Therapeutics. Stock solutions were made using DMSO and working stock solutions were made using RPMI 1640 media containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Akt1/2 kinase inhibitor (Akti) was purchased from Sigma. MTT assay was performed to study drug induced cytotoxicity and thymidine uptake was used as a measure to study differences in proliferation. Flow cytometry using Annexin V-FITC and propidium iodide (PI) was used to measure drug induced apoptosis in cell lines and patient cells. In addition, apo-2.7 was also used to measure apoptosis in patient cells. Mitocapture and cytochrome-c assays were also performed to confirm the induction of apoptosis in MM cell lines. In order to study the mechanism of action of the drug, immunoblotting studies were performed on lysates made from cell lines incubated with the drug for various time points. Results: AT219 induced potent cytotoxicity in MM cell lines MM1S, MM1R and H929, all three expressing wild type p53 with IC50 values of 2.5–5μM. Similar effects were observed when the above mentioned cell lines were treated with AT219 and the inhibitory effect of proliferation of these cells were examined. When MM1S or H929 cells were cultured with bone marrow stromal cells (BMSCs) derived from MM patients or with one of the three tumor promoting cytokines implicated in MM (IL6, IGF or VEGF) and treated with AT219, the drug was able to inhibit the proliferation of both cell lines to similar extents as observed when cultured independently without BMSCs or the cytokines. The increase in cytotoxicity was found to be due to cells undergoing apoptosis as observed when MM1S or H929 cells were cultured with AT219 and % apoptotic cells were measured as measured by annexin/PI, mitocapture and cytochrome c assays. AT219 was also observed to induce more potent apoptosis in primary cells obtained from new MM patients with wild type p53 than in cells obtained from relapsed MM patients with wild type p53. AT219 clearly upregulated p53 as observed by performing immunoblots after treatment with the drug in MM1S and H929 cells. In addition, MDM2 and p21 were also found to be significantly upregulated and Bax was slightly upregulated post drug treatment. Bcl2, Mcl1 and Xiap levels were down regulated. In MM1S cells AT219 treatment resulted in a slight down regulation of pAkt (Ser 473). However, in H929 cells we observed a transient upregulation of pAkt following AT219 treatment. This prompted us to test AT219 in combination with Akti on MM cell lines. Our results on both MM1S and H929 cells using AT219 in combination with Akti demonstrated synergy. We are currently testing this combination in primary cells drawn from MM patients with both wild type p53 and those with p53 deletions and mutations. Conclusions: Our studies validate the anti-MM activity of AT219 in MM patients with wild type p53. In addition to using AT219 in combination with Akti, we are testing AT219 in combination with existing anti- MM chemotherapeutic agents. Interesting results from our studies will form the basis for clinical evaluation of AT219 as a single agent or in combination with an Akt inhibitor or other agents in MM patients. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Merck: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; Novartis: Research Funding; Genzyme: Research Funding; Cephalon: Research Funding.

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In silico screening identifies a novel small molecule inhibitor that counteracts PARP inhibitor resistance in ovarian cancer

Scientific Reports ◽

10.1038/s41598-021-87325-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Z. Ping Lin ◽

Nour N. Al Zouabi ◽

Mark L. Xu ◽

Nicole E. Bowen ◽

Terence L. Wu ◽

...

Keyword(s):

Ovarian Cancer ◽

Small Molecule ◽

In Silico ◽

Parp Inhibitor ◽

Parp Inhibitors ◽

Small Molecule Inhibitor ◽

Wild Type ◽

In Silico Screening ◽

Molecule Inhibitor ◽

Inhibitor Resistance

AbstractPoly ADP-ribose polymerase (PARP) inhibitors are promising targeted therapy for epithelial ovarian cancer (EOC) with BRCA mutations or defective homologous recombination (HR) repair. However, reversion of BRCA mutation and restoration of HR repair in EOC lead to PARP inhibitor resistance and reduced clinical efficacy of PARP inhibitors. We have previously shown that triapine, a small molecule inhibitor of ribonucleotide reductase (RNR), impaired HR repair and sensitized HR repair-proficient EOC to PARP inhibitors. In this study, we performed in silico screening of small molecule libraries to identify novel compounds that bind to the triapine-binding pocket on the R2 subunit of RNR and inhibit RNR in EOC cells. Following experimental validation of selected top-ranking in silico hits for inhibition of dNTP and DNA synthesis, we identified, DB4, a putative RNR pocket-binding inhibitor markedly abrogated HR repair and sensitized BRCA-wild-type EOC cells to the PARP inhibitor olaparib. Furthermore, we demonstrated that the combination of DB4 and olaparib deterred the progression of BRCA-wild type EOC xenografts and significantly prolonged the survival time of tumor-bearing mice. Herein we report the discovery of a putative small molecule inhibitor of RNR and HR repair for combination with PARP inhibitors to treat PARP inhibitor-resistant and HR repair-proficient EOC.

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