Rooha Contractor

The Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase Inhibitor AZD6244 (ARRY-142886) Induces Growth Arrest in Melanoma Cells and Tumor Regression When Combined with Docetaxel

Purpose: Disseminated melanoma is highly therapy resistant. The finding that 66% of melanomas harbor the activating BRAFV600E mutation has raised expectations for targeting the Ras/RAF/mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway in melanoma. This study addresses the anti-melanoma activity of the MEK inhibitor AZD6244 (ARRY-142886). Experimental Design: We recently have shown that growing melanoma cells as three-dimensional collagen-implanted spheroids enhances resistance to the MEK inhibitor U0126. Here, we investigated the anti-melanoma activity of AZD6244 in two-dimensional cell culture, the three-dimensional spheroid model, and an in vivo model. Results: In two-dimensional cell culture, AZD6244 was cytostatic and reduced the growth of melanoma cells in a concentration-dependent fashion through the induction of G1-phase cell cycle arrest. In our three-dimensional spheroid model, the effects of AZD6244 were largely cytostatic and reversible, with drug washout leading to spheroid regrowth. Finally, 1205Lu cells were grown as tumor xenografts in severe combined immunodeficient mice. After tumor establishment, mice were dosed twice daily with 0, 10, or 30 mg/kg AZD6244 p.o. AZD6244 treatment decreased phospho-ERK in the tumors and significantly suppressed tumor growth. The original tumors remained viable, suggesting that AZD6244 monotherapy was largely cytostatic, and not proapoptotic in this model. Further studies showed that co-administration of AZD6244 (30 mg/kg) with docetaxel (15 mg/kg) led to tumor regression, indicating the potential for MEK inhibitor/chemotherapy drug combinations. Conclusions: Inhibition of MEK is cytostatic as a monotherapy in melanoma, but cytotoxic when combined with docetaxel.

Mechanisms of Antileukemic Activity of the Novel Bcl-2 Homology Domain-3 Mimetic GX15-070 (Obatoclax)

In this study, we investigated the mechanism of apoptosis induction of obatoclax (GX15-070), a novel Bcl-2 homology domain-3 (BH3) mimetic, in acute myeloid leukemia (AML) cell lines and primary AML samples. Obatoclax inhibited cell growth of HL-60, U937, OCI-AML3, and KG-1 cell lines. Apoptosis induction contributed to the observed antiproliferative effects at concentrations of this agent that mirror its affinity for antiapoptotic Bcl-2 proteins. We show that obatoclax can promote the release of cytochrome c from isolated leukemia cell mitochondria and that apoptosis induced by this agent is preceded by the release of Bak from Mcl-1, liberation of Bim from both Bcl-2 and Mcl-1, and the formation of an active Bak/Bax complex. Notably, apoptosis was diminished, but not fully prevented, in the absence of Bak/Bax or Bim, suggesting that obatoclax has additional targets that contribute to its cytotoxicity. At growth inhibitory doses that did not induce apoptosis or decrease viability, obatoclax induced an S-G(2) cell-cycle block. Obatoclax induced apoptosis in AML CD34+ progenitor cells with an average IC(50) of 3.59 +/- 1.23 micromol/L although clonogenicity was inhibited at concentrations of 75 to 100 nmol/L. Obatoclax synergized with the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells and synergistically induced apoptosis in combination with AraC in leukemic cell lines and in primary AML samples. In conclusion, we show that obatoclax potently induces apoptosis and decreases leukemia cell proliferation and may be used in a novel therapeutic strategy for AML alone and in combination with other targeted agents and chemotherapeutics.

An Organometallic Protein Kinase Inhibitor Pharmacologically Activates p53 and Induces Apoptosis in Human Melanoma Cells

Unlike other tumors, melanomas harbor wild-type (WT) p53 but exhibit impaired p53-dependent apoptosis. The mechanisms for the impaired p53 activation are poorly understood but may be linked to the high expression of the p53 suppressor Mdm2, which is found in >50% of melanoma lesions. Here, we describe an organometallic glycogen synthase kinase 3beta (GSK3beta) inhibitor (DW1/2) as a potent activator of p53 and inducer of cell death in otherwise highly chemoresistant melanoma cells. Using RNA interference and pharmacologic approaches, we show that p53 is required for the cytotoxic effects of this organometallic inhibitor. The DW1/2 compound was barely able to induce cell death in melanoma cells with p53 mutations, further confirming the requirement for p53-WT in the cytotoxic effects of the GSK3beta inhibition. Mechanistic analysis of the p53-dependent cell death indicated an apoptotic mechanism involving depolarization of mitochondrial membrane potential, caspase cleavage, and elevated NOXA expression. The effect of p53 was not simply due to passive up-regulation of protein expression as adenoviral-mediated overexpression of p53 was not able to induce cell death. Treatment of melanoma cells with DW1/2 was instead found to decrease levels of Mdm2 and Mdm4. The importance of Mdm2 down-regulation in DW1/2-induced apoptosis was confirmed by treating the p53-WT cells with the p53/Mdm2 antagonist Nutlin-3. Taken together, our data provide a new strategy for the pharmacologic activation of p53 in melanoma, which may be a viable approach for overcoming apoptotic resistance in melanoma and offer new hope for rational melanoma therapy.

Identification of a Novel Subgroup of Melanomas with KIT/Cyclin-Dependent Kinase-4 Overexpression

Although many melanomas harbor either activating mutations in BRAF or NRAS, there remains a substantial, yet little known, group of tumors without either mutation. Here, we used a genomic strategy to define a novel group of melanoma cell lines with co-overexpression of cyclin-dependent kinase 4 (CDK4) and KIT. Although this subgroup lacked any known KIT mutations, they had high phospho-KIT receptor expression, indicating receptor activity. Quantitative PCR confirmed the existence of a similar KIT/CDK4 subgroup in human melanoma samples. Pharmacologic studies showed the KIT/CDK4-overexpressing subgroup to be resistant to BRAF inhibitors but sensitive to imatinib in both in vitro and in vivo melanoma models. Mechanistically, imatinib treatment led to increased apoptosis and G(1) phase cell cycle arrest associated with the inhibition of phospho-ERK and increased expression of p27(KIP). Other melanoma cell lines, which retained some KIT expression but lacked phospho-KIT, were not sensitive to imatinib, suggesting that KIT expression alone is not predictive of response. We suggest that co-overexpression of KIT/CDK4 is a potential mechanism of oncogenic transformation in some BRAF/NRAS wild-type melanomas. This group of melanomas may be a subpopulation for which imatinib or other KIT inhibitors may constitute optimal therapy.

The novel triterpenoid CDDO-Me suppresses MAPK pathways and promotes p38 activation in acute myeloid leukemia cells

Development of novel therapeutic strategies is a continuing challenge for the treatment of acute myeloid leukemia (AML). The novel triterpenoid, C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid (CDDO-Me), induces apoptosis in myeloid leukemic cell lines and in primary AML samples. In this report, the effects of CDDO-Me on CD34+ AML progenitor cells in vitro were examined. CDDO-Me induced apoptosis in all but one of ten AML samples. CDDO-Me is known to inhibit the activation of ERK1/2. In this series of primary AML samples, ERK was expressed and phosphorylated in all patient samples studied and CDDO-Me inhibited ERK phosphorylation in five of 10 samples. However, CDDO-Me induced apoptosis in four of five samples without decreasing pERK levels, suggesting that pERK is not the sole target of the compound. CDDO-Me induced phosphorylation of p38 in AML-derived U937 cells. Pretreatment of U937 cells with a p38 inhibitor protected cells from the cyto-toxic effects of CDDO-Me. These findings suggest a role for p38 in CDDO-Me-induced apoptosis. In preliminary studies, CDDO-Me induced p38 phosphorylation in seven of eight primary AML samples. These findings suggest that CDDO-Me treatment shifts cell signaling away from cyto-protective pathways and thus CDDO-Me may be effective for the treatment of AML.

Novel role of HDAC inhibitors in AML1/ETO AML cells: activation of apoptosis and phagocytosis through induction of annexin A1

The chimeric fusion protein AML1-ETO, created by the t(8;21) translocation, recruits histone deacetylase (HDAC) to AML1-dependent promoters, resulting in transcriptional repression of the target genes. We analyzed the transcriptional changes in t(8;21) Kasumi-1 AML cells in response to the HDAC inhibitors, depsipeptide (FK228) and suberoylanilide hydroxamic acid (SAHA), which induced marked growth inhibition and apoptosis. Using cDNA array, annexin A1 (ANXA1) was identified as one of the FK228-induced genes. Induction of ANXA1 mRNA was associated with histone acetylation in ANXA1 promoter and reversal of the HDAC-dependent suppression of C/EBPα by AML1-ETO with direct recruitment of C/EBPα to ANXA1 promoter. This led to increase in the N-terminal cleaved isoform of ANXA1 protein and accumulation of ANXA1 on cell membrane. Neutralization with anti-ANXA1 antibody or gene silencing with ANXA1 siRNA inhibited FK228-induced apoptosis, suggesting that the upregulation of endogenous ANXA1 promotes cell death. FK228-induced ANXA1 expression was associated with massive increase in cell attachment and engulfment of Kasumi-1 cells by human THP-1-derived macrophages, which was completely abrogated with ANXA1 knockdown via siRNA transfection or ANXA1 neutralization. These findings identify a novel mechanism of action of HDAC inhibitors, which induce the expression and externalization of ANXA1 in leukemic cells, which in turn mediates the phagocytic clearance of apoptotic cells by macrophages.

Bcl2 phosphorylation and active PKC α are associated with poor survival in AML [13]

additional 203 bp PCR product. The second strategy consists of nested PCR, and subsequent restriction enzyme digestion with BsaXI (New England Biolabs, Hitchin, UK). In case of mutated DNA, the PCR product remains undigested, whereas unmutated DNA results in two fragments of 170 and 203 bp in size. None of the MDS/AML cases showed evidence of the JAK2 V617F mutation, regardless of the presence or absence of fibrosis. Interestingly, the mutation was detected in three of four cases (75%) diagnosed as MDS/MPD. In contrast to the MDS/AML with fibrosis, these four patients showed some clinical features of chronic myeloproliferative disease, including splenomegaly, leukocytosis and/or thrombocytosis, as well as, morphological features of CMPD in the trephine biopsy. One of the positive cases was classified as atypical chronic myelogenous leukemia negative for BCR/ABL fusion transcripts. In contrast to the results from Ohyashiki et al., our results inidicate that the JAK2 V617F mutation is exceedingly rare in bona fide MDS or de novo AML, regardless of the presence of fibrosis. Furthermore, the identification of the JAK2 mutation in MDS/MPD cases indicates that these cases are more closely related to classical CMPD. This interpretation is supported by other reports demonstrating the occurrence of the JAK2 mutation in MDS/MPD, such as chronic myelomonocytic leukemia, although the latter entity shows a lower incidence of JAK2 mutations than CMPD. We believe that the analysis of the JAK2 V617F mutation, in difficult-to-classify cases, will help to clarify the borderline between MDS, on one hand, and atypical CMPD and MDS/MPD on the other hand. The better understanding of these entities, potentially may lead in the near future to identify new therapeutic options for these patients.

PPARgamma-Active triterpenoid CDDO enhances ATRA-induced differentiation in APL

Acute promyelocytic leukemia (APL) is associated with oncogenic PML-RARα that acts as a dominant negative transcriptional repressor of retinoic acid (RA) receptor target genes by recruiting histone deacetylase (HDAC). The peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptor family that forms heterodimers with retinoid X receptor (RXR). In addition to RAR targets, PML-RARα silence a wide range of nuclear receptor target genes including PPARγ targets. All-trans-retinoic acid (ATRA), a ligand for the RA receptor (RAR), restores normal retinoid signaling and induces terminal differentiation of APL cells; however, APL cells can develop resistance to ATRA. Using ATRA sensitive NB4 and ATRA-resistant derivative NB4MR2 cell lines, we demonstrate that PPARγ ligand 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) enhances pro-apoptotic and differentiating effects of ATRA in ATRA-sensitive NB4 cells and partially reverses ATRA resistance in NB4MR2 cells. The CDDO/ATRA combination synergistically induces RARβ2 expression both in ATRA-sensitive and -resistant APL cells. RARβ2 mRNA induction by CDDO/ATRA was mediated in part by enhanced H3-Lys9 acetylation in the RARβ2 promoter which in turn increased the affinity of RARβ for βRARE. PPARγ specific inhibitor T007 and silencing of PPARγ by siRNA diminished CDDO-induced maturation and RARβ2 mRNA along with PPARγ induction indicating that PPARγ activation is at least partially responsible for the RARβ2 transcription and maturation induction. In an in vivo mouse model of APL, CDDO derivative CDDO-methyl ester markedly enhanced ATRA-induced maturation and extended the survival of mice. In summary, these results provide rationale for the combined targeting of RAR and PPARγ nuclear receptors in the therapy of APL.

PML-RARα and AML1–ETO translocations are rarely associated with methylation of the RARβ2 promoter

The acute promyelocytic leukemia-specific PML–RARα fusion protein is a dominant-negative transcriptional repressor of retinoic acid receptor (RAR) target genes, which recruits HDAC and corepressor proteins and inhibits coactivators. Another oncogenic transcription factor, AML1–ETO, was proposed to cause an HDAC-dependent repression of RAR target genes. The RAR target RARβ2 gene has been reported to be frequently silenced by hypermethylation in many types of cancer cells. We examined the methylation status of the RARβ2 and asked if demethylation could reverse ATRA resistance in ATRA-resistant PML–RARα and AML1–ETO-positive cells. PML–RARα positive NB4 and its ATRA-resistant subvariant MR2 and AML1–ETO expressing Kasumi-1 cells had heterozygous methylation of RARβ2. Although DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine partially reversed RARβ2 CpG methylation in these cells, it did not significantly enhance ATRA-induced RARβ2 mRNA expression and induction of maturation. However, the histone acetylase inhibitor SAHA combined with ATRA significantly reactivated RARβ2 mRNA both in NB4 and MR2 cells with degradation of PML–RARα, which was associated with maturation. In contrast, SAHA did not affect AML1–ETO levels and failed to induce RARβ2 expression and maturation in Kasumi-1 cells. In primary AML samples, RARβ2 expression was uniformly low; however, no specific correlation was observed between the methylation of the RARβ2 gene and expression of the fusion proteins, PML–RARα, and AML1–ETO. These results demonstrate that oncogenic PML–RARα and AML1–ETO translocations are rarely associated with RARβ2 promoter methylation in primary AML samples.

Development of a Conditional In vivo Model to Evaluate the Efficacy of Small Molecule Inhibitors for the Treatment of Raf-Transformed Hematopoietic Cells

Conditionally active forms of the Raf proteins (Raf-1, B-Raf, and A-Raf) were created by ligating NH2-terminal truncated activated forms (Delta) to the estrogen receptor (ER) hormone-binding domain resulting in estradiol-regulated constructs (DeltaRaf:ER). These different Raf:ER oncoproteins were introduced into the murine FDC-P1 hematopoietic cell line, and cells that grew in response to the three DeltaRaf:ER oncoproteins were isolated. The ability of FDC-P1, DeltaRaf-1:ER, DeltaA-Raf:ER, and DeltaB-Raf:ER cells to form tumors in severe combined immunodeficient mice was compared. Mice injected with DeltaRaf:ER cells were implanted with beta-estradiol pellets to induce the DeltaRaf:ER oncoprotein. Cytokine-dependent parental cell lines did not form tumors. Implantation of beta-estradiol pellets into mice injected with DeltaRaf:ER cells significantly accelerated tumor onset and tumor size. The recovered DeltaRaf:ER cells displayed induction of extracellular signal-regulated kinase (ERK) in response to beta-estradiol stimulation, indicating that they had retained conditional activation of ERK even when passed through a severe combined immunodeficient mouse. The DeltaRaf:ER cells were very sensitive to induction of apoptosis by the mitogen-activated protein/ERK kinase (MEK) 1 inhibitor CI1040 whereas parental cells were much less affected, demonstrating that the MEK1 may be useful in eliminating Ras/Raf/MEK-transformed cells. Furthermore, the effects of in vivo administration of the MEK1 inhibitor were evaluated and this inhibitor was observed to suppress the tumorigenicity of the injected cells. This DeltaRaf:ER system can serve as a preclinical model to evaluate the effects of signal transduction inhibitors which target the Raf and MEK proteins.