Using Small Molecules To Identify Critical Signaling Pathways Of Mutant N-RAS In Acute Leukemia Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 169-169
Author(s):  
Atsushi Nonami ◽  
Martin Sattler ◽  
Ellen L. Weisberg ◽  
Jianming Zhang ◽  
Qingsong Liu ◽  
...  
Keyword(s):  
Small Molecules ◽  
Signaling Pathways ◽  
Small Molecule ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Ras Signaling ◽  
Over Expression ◽  
Simultaneous Inhibition ◽  
Chemical Screen ◽  
Multiple Signaling

Abstract Activating point mutations in NRAS are detected in more than 10% of AML patients, making NRAS an important therapeutic target. Using small molecules to directly target NRAS or inhibit post-translational modification, such as farnesylation, have been extensively investigated. The potential of strategies focused on targeting downstream effectors of RAS, such as RAF or MEK, has been limited by the complexity of RAS signaling, including redundancy and feedback loops. Large-scale RNAi screens have been used to identify genes (TBK1, STK33 and GATA2, for example) that are synthetically lethal with RAS mutations and these are being explored as therapeutic targets. Recognizing the complexity of RAS signaling, we tested the notion that small molecule screens designed to simultaneously inhibit multiple signaling pathways might identify combinations of pathways that are critical for NRAS signaling in leukemic cells. Initially, we created an experimental Ba/F3 cell line model that was completely dependent on oncogenic N-RAS-G12D for growth and survival. Knockdown of NRAS suppressed growth >95%, but could be rescued by interleukin-3 (IL-3). A chemical screen using panels of multi-targeted small molecule kinase inhibitors against BaF3-NRAS-G12D cells revealed a lead compound, NRAS1 (N-(4-methyl-3-(1-methyl-7-(6-methylpyridin-3-ylamino)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)phenyl)-3-(trifluoromethyl)benzamide), with high selectivity and sensitivity toward leukemia cell lines with NRAS mutations in vitro. A number of studies were then performed to investigate the targets of this compound. Transcriptional profiling before and after treatment of two AML cell lines with NRAS mutation (OCI-AML3 and KO52 cells, respectively) showed profiles similar to that obtained by knocking down NRAS, supporting the hypothesis that this compound suppressed NRAS signaling. Biochemical studies demonstrated that NRAS1 did not inhibit several classical targets of RAS signaling, including, RAF, MEK and ERK. In contrast, NRAS1 was found to substantially reduce AKT and RPS6 phosphorylation. Over-expression of a constitutively active allele of AKT, myrAKT, in Ba/F3-NRAS G12D cells conferred strong resistance to NRAS1, confirming that suppression of phospho-AKT may be important for the function of NRAS1. However, direct inhibition of AKT only partially recapitulated the effects of NRAS1. Kinase selectivity profiling of NRAS1 (1μM) in OCI-AML3 cells (EC50: 0.3μM) identified 13 major binding partners with more than 85% efficacy. The targets consisted mainly of SRC family proteins (ie SRC, FGR, and LYN) and MAPK family proteins (ie GCK, KSH, and p38), but not MEK1/2, ERK1/2 or AKT1-3. A series of analogs of NRAS1 was synthesized and structure/function studies were carried out. One compound, (LKB-0304601, 1% EC50 of original compound) lost the ability to bind to the MAP4K family of proteins, especially GCK (MAPK4K2). A combination effect was observed between a known GCK inhibitor, NG25, and a known allosteric AKT inhibitor, MK-2206, against mutant NRAS-expressing cells. This finding supports the hypothesis that simultaneous inhibition of GCK and AKT has suppressive activity against leukemia cells transformed by NRAS. Furthermore, a putative gate-keeper mutation introduced into GCK (GCK G96S) resulted in partial resistance to growth suppression by NG25 or NRAS1. Growth suppression of NRAS-transformed leukemic cells was further induced by knock-down of GCK by shRNAs in cells with mutant NRAS, THP-1 cells and MOLT-3, and this effect could be rescued by over-expression of GCK. Finally, in a xenotransplant model using NRAS-mutant-expressing OCI-AML3 cells and MOLT-3 cells, NRAS1 significantly reduced tumor burden and prolonged survival compared to controls. Overall, by using a chemical screen designed to inhibit multiple signaling pathways simultaneously in oncogene-addicted cells, followed by signaling studies, cell biological studies and kinase selectivity profiling, we found that simultaneous inhibition of AKT and GCK, by either NRAS1 or selective inhibitors, exhibits activity against NRAS-transformed leukemia cells. Disclosures: Griffin: Novartis Pharmaceuticals: Research Funding.

scholarly journals Targeting Th17 Cells with Small Molecules and Small Interference RNA

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Hui Lin ◽  
Pingfang Song ◽  
Yi Zhao ◽  
Li-Jia Xue ◽  
Yi Liu ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Th17 Cells ◽  
Therapeutic Potential ◽  
Random Sequence ◽  
Orphan Receptor ◽  
T Helper 17 ◽  
Small Interference Rna ◽  
Chemical Screen ◽  
Interference Rna

T helper 17 (Th17) cells play a central role in inflammatory and autoimmune diseases via the production of proinflammatory cytokines interleukin- (IL-) 17, IL-17F, and IL-22. Anti-IL-17 monoclonal antibodies show potent efficacy in psoriasis but poor effect in rheumatoid arthritis (RA) and Crohn’s disease. Alternative agents targeting Th17 cells may be a better way to inhibit the development and function of Th17 cells than antibodies of blocking a single effector cytokine. Retinoic acid-related orphan receptor gamma t (RORγt) which acts as the master transcription factor of Th17 differentiation has been an attractive pharmacologic target for the treatment of Th17-mediated autoimmune disease. Recent progress in technology of chemical screen and engineering nucleic acid enable two new classes of therapeutics targeting RORγt. Chemical screen technology identified several small molecule specific inhibitors of RORγt from a small molecule library. Systematic evolution of ligands by exponential enrichment (SELEX) technology enabled target specific aptamers to be isolated from a random sequence oligonucleotide library. In this review, we highlight the development and therapeutic potential of small molecules inhibiting Th17 cells by targeting RORγt and aptamer mediated CD4+T cell specific delivery of small interference RNA against RORγt gene expression to inhibit pathogenic effector functions of Th17 lineage.

Screening Small Molecule Inhibitors of VLA-5 to Block the Interaction Between Acute Lymphoblastic Leukemia with Ph+ and Their Microenvironment.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1027-1027
Author(s):  
Zhongbo Hu ◽  
Xiaomiao Li ◽  
David Ostrov ◽  
William Slayton
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Small Molecules ◽  
Small Molecule ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Lymphoblastic Leukemia ◽  
Marrow Stromal Cells ◽  
Leukemia Cells ◽  
Human Fibronectin

Abstract Abstract 1027 Integrin VLA-5 (α5β1, CD49e/CD29) plays an important role in hematopoietic cells functioning as well as in promoting tumor angiogenesis and tumor metastasis. Molecules targeting VLA-5 can be rapidly developed into anti-inflammatory and anti-tumor pharmaceuticals. VLA-5 is highly expressed on Ph+ leukemia cells and VLA-5 inhibitory antibodies can significantly inhibit the adhesion of Ph+ leukemia cells to human fibronectin. We generated an atomic homology model of VLA-5 based on the crystal structure of the extracellular segment of integrin αVβ3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence and utilized this structure-based approach to identify VLA-5 binding drug-like small molecules. We selected the Arg-Gly-Asp binding residues and the epitopes of VLA-5 antibody as the target for small molecule binding using SPHERE_SELECT in DOCK6. The grid-based scoring system was used for scoring with the non-bonded force field energy function. The 100 highest scoring small molecules were assayed in an in vitro adhesion assay using leukemia cell lines and solid phase assay. This approach identified several leading small-molecule compounds, V10, V20, V37 and L4. Their IC50 are respectively 22.5μM, 23.7μM, 32.0μM and 28.9μM. These compounds can inhibit the adhesion of VLA-5 expressing Philadelphia chromosome positive leukemia to both human fibronectin and bone marrow stromal cells. Compounds V10 and V20 also significantly inhibited the growth of Ph+ leukemia cells. These compounds can enhance the effect of imatinib and dasatinib to kill Ph+ leukemia cells when cultured contacting with bone marrow stromal cells. We are currently testing the synergistic effect of these compounds with tyrosine kinase inhibitors to treat the Ph+ acute lymphoblastic leukemia in NOD/SCID animal model. Disclosures: No relevant conflicts of interest to declare.

Hyperactive Ptpn11 Mutations Alters Leukemic Stem Cell Frequency through Mcl-1 Overexpression

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3565-3565
Author(s):  
Lili Chen ◽  
Wei Chen ◽  
Maria Mysliwski ◽  
Justin Serio ◽  
James Ropa ◽  
...  
Keyword(s):  
Stem Cell ◽  
Small Molecule ◽  
Leukemic Cells ◽  
Receptor Protein ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Type I ◽  
Leukemic Stem Cell ◽  
Cell Frequency ◽  
Apoptotic Gene

Abstract Shp2 is a non-receptor protein-tyrosine phosphatase encoded by PTPN11 and implicated in the Ras, JAK-STAT and PI3K pathways. Activating mutations in Shp2 are found in patients with developmental disorders such as Noonan and LEOPARD syndrome, as well as, hematologic malignancies. Although rare in most other solid tumors, Shp2 mutations are common in juvenile myelomonocytic leukemia (JMML) accounting for ~35% of cases. To understand its role as a cooperating mutation in AML we sequenced PTPN11 in human samples. Here we report that Shp2 mutations are present in human AML at a rate of 6.6% (6/91) in the ECOG E1900 dataset. To investigate the biological function of Shp2 mutations we asked how this functions in a cooperative model of leukemogenesis with the MLL-AF9 fusion protein. Despite showing increased genetic stability compared to other leukemias, MLL leukemias commonly contain type I mutations that can functionally cooperate resulting in more aggressive leukemias. These mutations often occur in genes encoding components of the Ras pathway including mutually exclusive mutations of NRAS, KRAS, PTPN11 and NF1 and account for ~37% of MLL rearranged leukemias. However, the mechanisms of cooperation with MLL fusion proteins are poorly understood. We found that the Shp2E76K activating mutation commonly found in humans significantly accelerates MLL-AF9 mediated leukemogenesis. The E76K mutation results in structural changes that confer increased phosphatase activity to the Shp2 protein and increased Ras signaling. We attribute the MLL-AF9/Shp2E76K cooperation to a more rapid leukemic initiation as evidenced in colony formation assays using mouse bone marrow HSPCs. Cells transduced with MLL-AF9/Shp2E76K expanded faster than MLL-AF9 cells at early stages following transduction, indicating more efficient transformation of myeloid progenitors than MLL-AF9 alone. Cytokine independent growth is achieved in MLL-AF9 cells following expression of Shp2E76K through the constitutive activation of the IL3 signaling pathway and ERK phosphorylation. Importantly, addition of Shp2E76K significantly accelerated MLL-AF9 mediated acute myeloid leukemia in mice, indicating activated Shp2 cooperates with MLL-AF9 in vivo. In addition, leukemic stem cell frequency was increased by greater than 4 fold due to Shp2E76K expression. As Shp2 is reported to regulate an anti-apoptotic gene program, we investigated these in the context of MLL-AF9 leukemic cells with and without Shp2E76K. While Bcl2, BclXL and Mcl-1, were upregulated in Shp2E76K cells, Mcl-1 showed the highest upregulation in response to Shp2E76K. Further, expression of Mcl-1 with MLL-AF9 in colony assays phenocopies expression of Shp2E76K suggesting that, mechanistically, Shp2 mutations may cooperate through activation of an anti-apoptotic gene program, primarily through Mcl-1. Finally, we asked how leukemic cells bearing Shp2E76K would respond to small molecule inhibition of Mcl-1. MLL-AF9 leukemic cells expressing Shp2E76K were desensitized to small molecule mediated Mcl-1 inhibition consistent with increased Mcl-1 protein. These data were confirmed in human cells where U937 cells, which contain an activating Shp2 mutation, exhibited resistance to Mcl-1 inhibition compared to ML2 or K562 cell which both bear wild type Shp2. Together, these data suggest patients with hyperactive Shp2 signaling may respond poorly to drugs targeting Mcl-1 due to an overabundance of the protein. Disclosures No relevant conflicts of interest to declare.

scholarly journals High-Dimensional Single-Cell Proteomic Analysis Reveals Therapeutic Vulnerabilities in Relapsed/Refractory AML By Concomitant Analysis of Cell Surface Antigens, Signaling Pathways and Anti-Apoptotic Proteins

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 26-26
Author(s):  
Muharrem Muftuoglu ◽  
Zoe Alaniz ◽  
Duncan Mak ◽  
Angelique J. Lin ◽  
Jared K. Burks ◽  
...  
Keyword(s):  
Single Cell ◽  
Signaling Pathways ◽  
Research Funding ◽  
Expression Patterns ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
High Dimensional ◽  
Therapeutic Approaches ◽  
Car T ◽  
Immune Phenotypes

Background: The heterogeneous and adaptive nature of AML-associated genomic and proteomic landscape may account for disease relapse and poor prognosis, as therapy-associated selective pressure drives the emergence and expansion of AML clones with features different from those detected at diagnosis. The evolving focus is on single-cell analytical tools to fully capture the pathobiological heterogeneity of AML. We leveraged CyTOF to interrogate the proteomic heterogeneity in patients with R/R AML, which could potentially permit the design of rational combinatorial therapeutic approaches targeting vulnerabilities in these cells.. Methods: To dissect AML heterogeneity and its contribution to treatment failure, we designed a 51-parameter CyTOF panel and interrogated cellular hierarchy, major immune phenotypes, anti-apoptotic molecules, signaling pathways, exhaustion markers and attractive targets for CAR T-cell therapy in R/R AML (n:13). Results: First, we generated two-dimensional t-SNE maps and observed that the leukemic bone marrow compartment harbored immature (CD34+CD38- and CD34+CD38+) and mature leukemic blasts (CD33+CD34-) and major immune subsets. Constitutively active signaling pathways characterized by high levels of p-4EBP1, p-MEK1/2, p-S6 and p-AKT, marked immature and mature leukemia cells and comparative analysis revealed that monocytic blasts harbored more active signaling networks. The proportions of these subpopulations varied significantly across patients. We initially assessed the distribution of anti-apoptotic molecules across these leukemia compartments. Strikingly, Bcl-2 levels were considerably high within less-differentiated leukemic cell compartments and CD68 expressing leukemic blasts with monocytic differentiation had significantly lower levels of Bcl-2. This suggests that differentiated leukemic cells could preferentially survive under selection pressure of Bcl-2 inhibitors. On the contrary, we observed a trend towards higher Mcl-1 levels in differentiated leukemia cells. These findings provide a rationale for combining therapeutic modalities to target different leukemia subpopulations. Indeed, Bcl-2 and Mcl-1 inhibitors (Venetoclax and AZD5991) resulted in highly synergistic effects in AML PDX models. Hence, this analysis supports the hypothesis that Mcl-1 overexpression is a resistance factor to Bcl-2 inhibition, usually understood as developing in the same cell. Next, we assessed expression patterns of putative CAR T-cell targets expressed on leukemic cells and identified significant variegated expression patterns in R/R AML samples. CD123 demonstrated patchy distribution across immature and differentiated leukemic blasts while CD33 expression was the main characteristic of differentiated leukemic blasts. Of note, the immature leukemia compartment demonstrated variable levels of CD33 and complete lack of CD33 on CD34+ leukemic cells was observed in a subgroup of patients. CLL-1 was uniformly expressed across all leukemia compartments, but was not ubiquitously expressed in all patient samples, revealing substantial interpatient heterogeneity in R/R AML and highlighting the concept of targeting at least two antigens concomitantly. Lastly, we sought to undertake high-dimensional assessment of immune compartments to identify major immune phenotypes in heavily treated R/R AML patients and discover the link between immune phenotypes and AML-associated traits. In line with leukemia cell heterogeneity, we found a significant degree of variation in immune cell composition among patients. Inhibitory molecules PD-1 and TIGIT were significantly expressed on CD4 and CD8 T-cells respectively, providing a rationale for use of combinatorial immunotherapeutic approach for the treatment of AML. Conclusion: Single-cell profiling of R/R AML using CyTOF reveals significantly heterogeneous expression patterns of molecules targeted by BH3 mimetics (Bcl-2 and Mcl-1), CAR T-cells, and other antibody-based immunotherapeutic therapies. This approach provides a rationale to develop combinatorial therapeutic approaches targeting distinct leukemia sub-populations with discrete expression patterns of established and novel putative targets. An example is the combined targeting of Bcl-2 and Mcl-1, which are differentially expressed in early and more differentiated leukemia subpopulations. Disclosures Carter: AstraZeneca: Research Funding; Syndax: Research Funding; Amgen: Research Funding; Ascentage: Research Funding. Andreeff:Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Amgen: Research Funding.

scholarly journals Reduced Expression of Sprouty1 Contributes to the Aberrant Proliferation and Impaired Apoptosis of Acute Myeloid Leukemia Cells

2019 ◽  
Author(s):  
Valentina Rosso ◽  
Crisitina Panuzzo ◽  
Jessica Petiti ◽  
Sonia Carturan ◽  
Matteo Dragani ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Tyrosine Kinases ◽  
Myeloid Leukemia ◽  
Ectopic Expression ◽  
Negative Regulator ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Ras Signaling ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

In most of acute myeloid leukemia patients there is an aberrant tyrosine kinases activity. The Sprouty family proteins were originally identified in Drosophila melanogaster as antagonists of Breathless, the mammalian ortholog of fibroblast growth factor receptor. This family proteins are inhibitors of RAS signaling induced by tyrosine kinases receptors and they are implicated in negative feedback processes regulating several intracellular pathways. The present study aims to investigate the role of a member of the Sprouty family, Sprouty1, as regulator of cell proliferation and growth in patients affected by acute myeloid leukemia. Sprouty1 mRNA and protein were both significantly down-regulated in acute myeloid leukemia cells compared to the normal counterpart, but they were restored when remission is achieved after chemotherapy. Ectopic expression of Sprouty1 revealed that it plays a key role in the proliferation and apoptotic defect that represent a landmark of the leukemic cells. Our study identified Sprouty1 as negative regulator involved in the aberrant signals of acute myeloid leukemia. Furthermore, we found a correlation between Sprouty1 and FoxO3a delocalization in AML at diagnosis, suggesting a multistep regulation of RAF–MEK–ERK signaling in human cancers. 

scholarly journals A novel drug-combination screen in zebrafish identifies epigenetic small molecule candidates for Duchenne muscular dystrophy

2020 ◽  
Author(s):  
Gist H. Farr ◽  
Melanie Morris ◽  
Arianna Gomez ◽  
Thao Pham ◽  
Elizabeth U. Parker ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Small Molecules ◽  
Small Molecule ◽  
Histone Deacetylase Inhibitors ◽  
Neuromuscular Disorder ◽  
Small Molecule Screen ◽  
Chemical Screen ◽  
Novel Drug

SummaryDuchenne muscular dystrophy (DMD) is a severe neuromuscular disorder and is one of the most common muscular dystrophies. There are currently few effective therapies to treat the disease, although many small-molecule approaches are being pursued. Specific histone deacetylase inhibitors (HDACi) can ameliorate DMD phenotypes in mouse and zebrafish animal models and have also shown promise for DMD in clinical trials. However, beyond these HDACi, other classes of epigenetic small molecules have not been broadly and systematically studied for their benefits for DMD. Here, we performed a novel chemical screen of a library of epigenetic compounds using the zebrafish dmd model. We identified candidate pools of epigenetic compounds that improve skeletal muscle structure in dmd zebrafish. We then identified a specific combination of two drugs, oxamflatin and salermide, that significantly rescued dmd zebrafish skeletal muscle degeneration. Furthermore, we validated the effects of oxamflatin and salermide in an independent laboratory. Our results provide novel, effective methods for performing a combination small-molecule screen in zebrafish. Our results also add to the growing evidence that epigenetic small molecules may be promising candidates for treating DMD.

ASPP2 Expression In Acute Leukemia Modulates Response to Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3143-3143
Author(s):  
Kerstin M Kampa-Schittenhelm ◽  
Charles D Lopez ◽  
Marcus Schittenhelm
Keyword(s):  
Tumor Suppressor ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Induction Chemotherapy ◽  
Small Molecule ◽  
Response To Therapy ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
P53 Pathway

Abstract Abstract 3143 Acute myeloid leukemias (AML) remain difficult to treat and therapy outcome is far from satisfactory for most disease subgroups. Inactivation of the p53 tumor suppressor pathway by mutation is a frequent event in many cancers that promotes tumorigenesis and resistance to chemotherapy. Although p53 mutations are rare in AML it is not well studied whether the p53 pathway is influenced by other mechanisms instead. ASPP2 (Apoptosis Stimulating Protein of p53 2) is a highly regulated member of a family of p53-binding proteins that enhance apoptosis at least in part through stimulation of p53-transactivation of selected pro-apoptotic target genes. We previously demonstrated in a mouse model that ASPP2 is a haploinsufficient tumor suppressor (Kampa et al., PNAS 2009), and low ASPP2 expression levels have been associated with aggressive courses of different tumors such as breast cancer and lymphoma. We have now studied how ASPP2 expression correlates with response to therapy using in vitro models as well as patient-derived acute leukemia cells collected pre- and post- chemotherapy. We first analyzed changes in ASPP2 protein expression after treatment with chemotherapy or small molecule tyrosine-kinase inhbitors. We found that ASPP2-induction was cell-type specific in various established leukemia lines including Jurkat and HL60 (ASPP2 levels induced) and MOLM14 cells (ASPP2 levels unchanged). To test if ASPP2 levels modulated growth or response to therapy of leukemia cells, we generated three different ASPP2-siRNA constructs and transiently introduced them by lipofection into various lymphoid and myelogenous leukemia cell lines, including K562, Kasumi1, HL60, MOLM14 and Jurkat. After ASPP2 silencing, we observed, with the exception of MOLM14 cells, an up to 3-fold increase in cell proliferation measured by an XTT-assay compared to an empty vector control. We also treated siRNA-silenced K562, Kasumi1, HL60 and Jurkat cell lines with daunorubicin or small molecules targeting cell line-specific mutations in FLT3, KIT or ABL. Cell lines with attenuated ASPP2 expression displayed a significantly (∼50%) lower rate of apoptosis-induction in AnnexinV-assays after chemotherapy as well as small molecule inhibitor treatment as compared to a negative control. Interestingly, treated and siRNA-silenced leukemic cells frequently demonstrated enlarged morphology consistent with mitotic catastrophe. To study ASPP2 expression in humans, we quantified ASPP2 levels by qRT-PCR and intracellular immunophenotyping in circulating leukemic cells derived from patients at several timepoints before and during induction-chemotherapy (n=63). We found that pre-treatment ASPP2 basal levels were variable in acute leukemias. Additionally, we found that induction-chemotherapy increased ASPP2 expression in leukemic cells in a subgroup of patients. Univariate and multivariate analysis of the correlation of available clinical data and patient outcomes with ASPP2 expression in these patient datasets is ongoing. Taken together, our results demonstrate that dysfunctional regulation of ASPP2 expression may contribute to the biology of leukemogenesis and to primary therapy resistance in a subgroup of patients with acute leukemia. This data provides important and clinically relevant insight into how the p53 pathway can be inactivated in acute leukemia and opens new avenues for investigation. Prospective clinical studies are warranted in order to further define the role of the ASPP2 pathway as a therapeutic target and as biomarker for response to therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Small Molecule Inhibitors of the Candida albicans Budded-to-Hyphal Transition Act through Multiple Signaling Pathways

PLoS ONE ◽  
2011 ◽  
Vol 6 (9) ◽  
pp. e25395 ◽  
Author(s):  
John Midkiff ◽  
Nathan Borochoff-Porte ◽  
Dylan White ◽  
Douglas I. Johnson
Keyword(s):  
Candida Albicans ◽  
Signaling Pathways ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Multiple Signaling

Deciphering the Critical Pathways of Mutant N-RAS in AML Using Small Molecule Inhibitors.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2455-2455
Author(s):  
Atsushi Nonami ◽  
Martin Sattler ◽  
Ellen L. Weisberg ◽  
Liu Qingsong ◽  
Jianming Zhang ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Small Molecules ◽  
Cell Line ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Small Gtpase ◽  
Ras Signaling ◽  
Chemical Screen

Abstract Abstract 2455 Activating mutations in the small GTPase N-RAS occur in about 10% of acute myeloid leukemia (AML) cases. Active N-RAS is thought to drive the disease and is therefore a potential target for drug development. There have been numerous unsuccessful efforts to target RAS itself with small molecules, and blocking post-translational modifications of RAS proteins, such as through inhibition of farnesyl transferase, has similarly not proven useful. In addition, efficacy of targeting critical downstream effectors has been limited by the complexity of RAS signaling, such as redundancy of signaling pathways and feedback mechanisms. While targeting RAS is challenging, it was our hypothesis that inhibiting the right combination of downstream pathways in a particular lineage with small molecules could be effective. Initially, we created a Ba/F3 cell line that was completely dependent on oncogenic N-RAS-G12D for growth and survival. Growth was suppressed >99% by shRNA for N-RAS, but could be rescued entirely by interleukin-3 (IL-3), which does not require N-RAS signaling in these cells. Using this cell line, we performed a high-throughput chemical screen with a large library of multi-targeted kinase inhibitors. The lead compound (NRAS1) showed a 70-fold difference in the EC50 for growth inhibition between BaF3-NRAS G12D cells cultured in the absence (0.01μM) or presence (0.77μM) of IL-3. Importantly, this compound showed selectivity towards several leukemia cell lines that were shown to be dependent on mutant N-RAS by shRNA compared to cells expressing wild-type N-RAS (p=0.02). Also, in a xenotransplant model using NRAS-G12D+ OCI-AML3 cells, this compound significantly reduced tumor burden (P=0.005) and prolonged survival (P=0.002) compared to controls. Next, we sought to identify the targets of NRAS1, Interestingly, the compound did not suppress MEK or ERK, which are classical targets of RAS signaling in epithelial cells. NRAS1 profoundly reduced AKT and RPS6 phosphorylation. Kinase selectivity profiling of this compound (1μM) in OCI-AML3 cells (EC50: 0.3μM) identified 13 major binding partners with more than 85% efficacy. The targets consisted mainly of SRC family proteins (SRC, FGR, and LYN etc.) and MAPK family proteins (MAP4K2, 3, 5, and p38 etc.) and others (ZAK and BTK etc), but not MEK and ERK, and AKT was not detected in this assay. In preliminary studies, most of these target kinases were knocked-down by shRNA and, as expected, no single kinase was found to be responsible for mediating growth inhibition. Using a phospho-antibody microarray, the most significantly de-phosphorylated kinases were p38, AKT and SRC, which supports our preliminary findings. To validate the significance of these results, we treated Ba/F3-N-RAS cells with combinations of kinase inhibitors. Combining the AKT inhibitor MK2206 and Dasatinib (SRC family inhibitor) revealed marked synergy, while neither had activity individually. Also, the combination of MK2206 and a cleaner SRC family inhibitor, AZD0530, also synergized, although to lesser extent. In both examples, however, the inhibition of N-RAS transformed cells by NRAS1 proved superior, suggesting that one or more additional targets are required for inhibition of NRAS signaling. To identify additional critical targets of our compound we generated several derivatives with different potency. In particular, one less potent analog of NRAS1 (analog 6, 1% EC50 of original compound) showed a loss of binding activity towards the MAP4K family of proteins, especially MAP4K2. Observed synergy between the selective MAP4K2 inhibitor NG25 and selective inhibitors of MK2206 and Dasatinib in Ba/F3-NRAS G12D cells further points toward MAP4K2 as being of additional significance for oncogenic RAS signaling. Together with the previous data, we propose AKT and MAP4K2 as critical targets of NRAS1. In conclusion, we have identified a novel and selective kinase inhibitor of the N-RAS signaling pathway by chemical screen using Ba/F3-N-RAS G12D cells. By combination of signaling study, kinase selectivity profiling and phosphoproteomics, the main functional targets were found to be AKT, and MAP4K2, and additional functional targets will be elucidated. Our approach also could be applied for other type of oncogenes, and it could help to find therapeutic compound and also help to decipher signaling mechanisms of the oncogenes which are thus far undruggable. Disclosures: No relevant conflicts of interest to declare.

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