Liat Goldberg

A Ras Inhibitor Tilts the Balance between Rac and Rho and Blocks Phosphatidylinositol 3-Kinase–Dependent Glioblastoma Cell Migration

Glioblastoma multiforme are highly aggressive tumors for which no adequate treatment has yet been developed. Glioblastoma multiforme show large amounts of active Ras, considered an appropriate target for directed therapy. Here, we show that the Ras inhibitor S-trans, trans-farnesyl thiosalicylic acid (FTS) can avert the transformation of human glioblastoma multiforme cells by inhibiting both their migration and their anchorage-independent proliferation. FTS, by down-regulating Ras activity in glioblastoma multiforme cells, inhibited phosphatidylinositol 3-kinase signaling, resulting in decreased activity of Rac-1. At the same time, activation of RhoA was increased. These two small GTPases are known to control the arrangement of the actin cytoskeleton. By tilting the balance between Rac-1 and RhoA activities, FTS caused the glioblastoma multiforme cells to undergo profound changes in morphology, including rearrangement of actin into stress fibers and assembly of focal adhesions, both of which are governed by RhoA signaling. These morphologic changes allowed strong attachment of the cells to the matrix, rendering them immobile. The results show that FTS should be considered as a candidate drug for glioblastoma multiforme therapy because it targets not only cell proliferation but also cell migration and invasion, which together constitute the most problematic aspect of these malignancies.

Identification of Regulators of Polyploidization Presents Therapeutic Targets for Treatment of AMKL

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.

Genome-scale expression and transcription factor binding profiles reveal therapeutic targets in transgenic ERG myeloid leukemia

The ETS transcription factor ERG plays a central role in definitive hematopoiesis, and its overexpression in acute myeloid leukemia (AML) is associated with a stem cell signature and poor prognosis. Yet how ERG causes leukemia is unclear. Here we show that pan-hematopoietic ERG expression induces an early progenitor myeloid leukemia in transgenic mice. Integrated genome-scale analysis of gene expression and ERG binding profiles revealed that ERG activates a transcriptional program similar to human AML stem/progenitor cells and to human AML with high ERG expression. This transcriptional program was associated with activation of RAS that was required for leukemia cells growth in vitro and in vivo. We further show that ERG induces expression of the Pim1 kinase oncogene through a novel hematopoietic enhancer validated in transgenic mice and human CD34(+) normal and leukemic cells. Pim1 inhibition disrupts growth and induces apoptosis of ERG-expressing leukemic cells. The importance of the ERG/PIM1 axis is further underscored by the poorer prognosis of AML highly expressing ERG and PIM1. Thus, integrative genomic analysis demonstrates that ERG causes myeloid progenitor leukemia characterized by an induction of leukemia stem cell transcriptional programs. Pim1 and the RAS pathway are potential therapeutic targets of these high-risk leukemias.

MicroRNA-486-5p is an erythroid oncomiR of the myeloid leukemias of Down syndrome.

Children with Down syndrome (DS) are at increased risk for acute myeloid leukemias (ML-DS) characterized by mixed megakaryocytic and erythroid phenotype and by acquired mutations in the GATA1 gene resulting in a short GATA1s isoform. The chromosome 21 microRNA (miR)-125b cluster has been previously shown to cooperate with GATA1s in transformation of fetal hematopoietic progenitors. In this study, we report that the expression of miR-486-5p is increased in ML-DS compared with non-DS acute megakaryocytic leukemias (AMKLs). miR-486-5p is regulated by GATA1 and GATA1s that bind to the promoter of its host gene ANK1. miR-486-5p is highly expressed in mouse erythroid precursors and knockdown (KD) in ML-DS cells reduced their erythroid phenotype. Ectopic expression and KD of miR-486-5p in primary fetal liver hematopoietic progenitors demonstrated that miR-486-5p cooperates with Gata1s to enhance their self renewal. Consistent with its activation of AKT, overexpression and KD experiments showed its importance for growth and survival of human leukemic cells. Thus, miR-486-5p cooperates with GATA1s in supporting the growth and survival, and the aberrant erythroid phenotype of the megakaryocytic leukemias of DS.

Perturbation of fetal hematopoiesis in a mouse model of Down syndrome’s transient myeloproliferative disorder

Children with Down syndrome develop a unique congenital clonal megakaryocytic proliferation disorder (transient myeloproliferative disorder [TMD]). It is caused by an expansion of fetal megakaryocyte-erythroid progenitors (MEPs) triggered by trisomy of chromosome 21 and is further enhanced by the somatic acquisition of a mutation in GATA1. These mutations result in the expression of a short-isoform GATA1s lacking the N-terminal domain. To examine the hypothesis that the Hsa21 ETS transcription factor ERG cooperates with GATA1s in this process, we generated double-transgenic mice expressing hERG and Gata1s. We show that increased expression of ERG by itself is sufficient to induce expansion of MEPs in fetal livers. Gata1s expression synergizes with ERG in enhancing the expansion of fetal MEPs and megakaryocytic precursors, resulting in hepatic fibrosis, transient postnatal thrombocytosis, anemia, a gene expression profile that is similar to that of human TMD and progression to progenitor myeloid leukemia by 3 months of age. This ERG/Gata1s transgenic mouse model also uncovers an essential role for the N terminus of Gata1 in erythropoiesis and the antagonistic role of ERG in fetal erythroid differentiation and survival. The human relevance of this finding is underscored by the recent discovery of similar mutations in GATA1 in patients with Diamond-Blackfan anemia.

E2F1 identified by promoter and biochemical analysis as a central target of glioblastoma cell-cycle arrest in response to ras inhibition

Active Ras contributes to the malignant phenotype of glioblastoma multiforme. Recent studies showed that the Ras inhibitor farnesyl thiosalicylic acid downregulates the transcription factor hypoxia‐inducible factor‐1α, causing shutdown of glycolysis in U87 glioblastoma cells. Farnesyl thiosalicylic acid also inhibited the growth of U87 cells. The way in which Ras inhibition affects U87 cell proliferation was not clear. Here we applied a computational method in which gene expression profile clustering is combined with promoter sequence analysis to obtain global dissection of the transcriptional response to farnesyl thiosalicylic acid in U87 cells. The analysis revealed a prominent Ras‐dependent cell‐cycle arrest response, in which a major component is highly enriched for the binding‐site signature of the transcription factor E2F1. Electrophoretic mobility shift assays together with E2F‐luciferase reporter assays showed that E2F1 was inactivated by the Ras inhibitor. Inhibition of Ras by farnesyl thiosalicylic acid promoted proteasomal degradation of cyclin D1, with a concomitant decrease in phosphorylated retinoblastoma protein accompanied by downregulation of E2F1 and decreased expression of key E2F1‐regulated genes critical for cell‐cycle progression. U87 cell growth arrest induced by farnesyl thiosalicylic acid was overridden by constitutive expression of E2F1. Thus, downregulation of E2F1 and of hypoxia‐inducible factor‐1α represents 2 distinct arms of the antioncogenic effect of Ras inhibitors in glioblastoma.

Salirasib (farnesyl thiosalicylic acid) for brain tumor treatment: a convection-enhanced drug delivery study in rats

Our aim was to assess the ability of convection-enhanced drug delivery (CED), a novel approach of direct delivery of drugs into brain tissue and brain tumors, to treat brain tumors using salirasib (farsnesyl thiosalicylic acid). CED was achieved by continuous infusion of drugs via intracranial catheters, thus enabling convective distribution of high drug concentrations over large volumes while avoiding systemic toxicity. Several phase II/III CED-based trials are currently in progress but have yet to overcome two major pitfalls of this methodology (the difficulty in attaining efficient CED and the significant nonspecific neurotoxicity caused by high drug doses in the brain). In this study, we addressed both issues by employing our previously described novel CED imaging and increased efficiency methodologies to exclusively target the activated form of the Ras oncogene in a 9L gliosarcoma rat model. The drug we used was salirasib, a highly specific Ras inhibitor shown to exert its suppressive effects on growth and migration of proliferating tumor cells in in vitro and in vivo models, including human glioblastoma, without affecting normal tissues. The results show a significant decrease in tumor growth rate in salirasib-treated rats relative to vehicle-treated rats as well as a significant correlation between CED efficacy and tumor growth rate with no observed toxicity despite drug concentrations an order of magnitude higher than previously detected in the brain. The results show that CED of salirasib is efficient and nontoxic for the treatment of glioblastoma in a rat model, thus suggesting that it may be considered for clinical application. [Mol Cancer Ther 2008;7(11):3609–16]

Overexpression of ERG in cord blood progenitors promotes expansion and recapitulates molecular signatures of high ERG leukemias

High expression of the ETS family transcription factor ERG is associated with poor clinical outcome in acute myeloid leukemia (AML) and acute T-cell lymphoblastic leukemia (T-ALL). In murine models, high ERG expression induces both T-ALL and AML. However, no study to date has defined the effect of high ERG expression on primary human hematopoietic cells. In the present study, human CD34+ cells were transduced with retroviral vectors to elevate ERG gene expression to levels detected in high ERG AML. RNA sequencing was performed on purified populations of transduced cells to define the effects of high ERG on gene expression in human CD34+ cells. Integration of the genome-wide expression data with other data sets revealed that high ERG drives an expression signature that shares features of normal hematopoietic stem cells, high ERG AMLs, early T-cell precursor-ALLs and leukemic stem cell signatures associated with poor clinical outcome. Functional assays linked this gene expression profile to enhanced progenitor cell expansion. These results support a model whereby a stem cell gene expression network driven by high ERG in human cells enhances the expansion of the progenitor pool, providing opportunity for the acquisition and propagation of mutations and the development of leukemia.

Somatic mutations in murine models of leukemia and lymphoma: Disease specificity and clinical relevance

Malignant transformation is a multistep process that is dictated by the acquisition of multiple genomic aberrations that provide growth and survival advantage. During the post genomic era, high throughput genomic sequencing has advanced exponentially, leading to identification of countless cancer associated mutations with potential for targeted therapy. Mouse models of cancer serve as excellent tools to examine the functionality of gene mutations and their contribution to the malignant process. However, it remains unclear whether the genetic events that occur during transformation are similar in mice and humans. To address that, we chose several transgenic mouse models of hematopoietic malignancies and identified acquired mutations in these mice by means of targeted re‐sequencing of known cancer‐associated genes as well as whole exome sequencing. We found that mutations that are typically found in acute myeloid leukemia or T cell acute lymphoblastic leukemia patients are also common in mouse models of the respective disease. Moreover, we found that the most frequent mutations found in a mouse model of lymphoma occur in a set of epigenetic modifier genes, implicating this pathway in the generation of lymphoma. These results demonstrate that genetically engineered mouse models (GEMM) mimic the genetic evolution of human cancer and serve as excellent platforms for target discovery and validation.