Gamil R. Antoun

The Human Glutathione S-Transferase P1 Protein Is Phosphorylated and Its Metabolic Function Enhanced by the Ser/Thr Protein Kinases, cAMP-Dependent Protein Kinase and Protein Kinase C, in Glioblastoma Cells

We report here that the human glutathione S-transferase P1 (GSTP1) protein, involved in phase II metabolism of many carcinogens and anticancer agents and in the regulation of c-Jun NH2-terminal kinase-mediated cell signaling, undergoes phosphorylation by the Ser/Thr protein kinases, cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), resulting in a significant enhancement of its metabolic activity. GSTP1 phosphorylation by PKA was glutathione (GSH)-dependent, whereas phosphorylation by PKC did not require but was significantly enhanced by GSH. In the presence of GSH, the stoichiometry of phosphorylation was 0.4 ± 0.03 and 0.53 ± 0.02 mol incorporated phosphate per mole of dimeric GSTP1 protein. The GSTP1 protein was phosphorylated, in the presence of GSH, by eight different PKC isoforms (α, βΙ, βΙΙ, δ, ε, γ, η, and ζ), belonging to the three major PKC subclasses, albeit with various efficiencies. The catalytic efficiency, kcat/Km, of the phosphorylated GSTP1 was more than double that of the unphosphorylated protein. In MGR3 human glioblastoma cells, PKA and PKC activation resulted in a significant increase in the level of phosphorylation of the GSTP1 protein and was accompanied by a 2.1- and 2.7-fold increase, respectively, in specific GSTP1 activity in the cells. Peptide phosphorylation analyses and both phosphorylation and enzyme kinetic studies with GSTP1 proteins mutated at candidate amino acid residues established Ser-42 and Ser-184 as putative phospho-acceptor residues for both kinases in the GSTP1 protein. Together, these findings show PKA- and PKC-dependent phosphorylation as a significant post-translational mechanism of regulation of GSTP1 function. The GSH-dependence of the phosphorylation suggests that under high intracellular GSH conditions, such as is present in most drug-resistant tumors, the GSTP1 protein will exist in a hyper-phosphorylated and enzymatically more active state. In normal cells, the functional activation of the GSTP1 protein by PKA- and PKC-dependent phosphorylation could represent a potentially important mechanism of cellular protection, whereas in tumors, increased phase II metabolism of anticancer drugs by the more active phosphorylated GSTP1 protein could contribute to the drug resistance and therapeutic failure frequently associated with increased activities of these Ser/Thr kinases.

Mitogen-activated protein kinase (MAPK) hyperactivation and enhanced NRAS expression drive acquired vemurafenib resistance in V600E BRAF melanoma cells.

Background: The response to vemurafenib in V600E BRAF+ve melanoma is short lived due to acquisition of vemurafenib resistance. Results: NRAS expression and increased MAPK activation drive vemurafenib resistance in V600E BRAF+ve melanoma. Conclusion: Resistance to vemurafenib in melanoma is complex and can be mitigated by MAPK and NRAS inhibition. Significance: These findings could lead to improved therapy of V600E BRAF+ve melanoma by targeting MAPKs and NRAS. Although targeting the V600E activating mutation in the BRAF gene, the most common genetic abnormality in melanoma, has shown clinical efficacy in melanoma patients, response is, invariably, short lived. To better understand mechanisms underlying this acquisition of resistance to BRAF-targeted therapy in previously responsive melanomas, we induced vemurafenib resistance in two V600E BRAF+ve melanoma cell lines, A375 and DM443, by serial in vitro vemurafenib exposure. The resulting approximately 10-fold more vemurafenib-resistant cell lines, A375rVem and D443rVem, had higher growth rates and showed differential collateral resistance to cisplatin, melphalan, and temozolomide. The acquisition of vemurafenib resistance was associated with significantly increased NRAS levels in A375rVem and D443rVem, increased activation of the prosurvival protein, AKT, and the MAPKs, ERK, JNK, and P38, which correlated with decreased levels of the MAPK inhibitor protein, GSTP1. Despite the increased NRAS, whole exome sequencing showed no NRAS gene mutations. Inhibition of all three MAPKs and siRNA-mediated NRAS suppression both reversed vemurafenib resistance significantly in A375rVem and DM443rVem. Together, the results indicate a mechanism of acquired vemurafenib resistance in V600E BRAF+ve melanoma cells that involves increased activation of all three human MAPKs and the PI3K pathway, as well as increased NRAS expression, which, contrary to previous reports, was not associated with mutations in the NRAS gene. The data highlight the complexity of the acquired vemurafenib resistance phenotype and the challenge of optimizing BRAF-targeted therapy in this disease. They also suggest that targeting the MAPKs and/or NRAS may provide a strategy to mitigate such resistance in V600E BRAF+ve melanoma.

DNA methyltransferase levels and altered CpG methylation in the total genome and in the GSTP1 gene in human glioma cells transfected with sense and antisense DNA methyltransferase cDNA.

This study examines the efficacy of using plasmid expression vectors containing sense and antisense DNA MTase cDNA to both up‐ and downregulate intracellular DNA MTase levels in human glioma cells. The effects of the changes in MTase levels on global genomic DNA methylation and on the methylation status of CpG dinucleotides in the GSTP1 gene were determined in a glioma cell line that overexpresses the GSTP1 gene. In cells transfected with sense DNA MTase cDNA, MTase gene transcripts increased to a maximum of 2.5‐fold at 24 h, while MTase activity increased to a maximum of 3.6‐fold at 48 h. The effects of antisense MTase cDNA transfections were less pronounced, and levels of MTase gene transcripts and enzyme activity in transfectants were decreased to only, approximately, one‐half the levels of controls. The alterations in DNA MTase expression were associated with corresponding changes in the level of global DNA methylation and in the methylation of the GSTP1 gene in the cells, however, with no detectable morphological or cytotoxic effects on the cells. No significant changes in GSTP1 gene expression were detected after the transfections, presumably because of the high levels of basal GSTP1 expression in the cells. Consequently, the p16 gene, known to be repressed transcriptionally by DNA methylation, was examined for the functional effects of the altered MTase levels. The results showed a 2‐fold decrease in p16 gene transcripts with the sense MTase transfectants, while in the MTase antisense‐transfected cells p16 transcript levels increased by 30%. Together, these results demonstrate the feasibility of using both sense and antisense DNA MTase expression vectors to regulate DNA MTase levels in glioma cells and that, over relatively short periods of time, the alterations in MTase activities are not deleterious to the cells. The system provides a model with which the role of DNA methylation in critical genes and DNA sequences can be investigated in glioma cells. J. Cell. Biochem. 77:372–381, 2000.

Molecular genetic evidence for a differentiation-proliferation coupling during DMSO-induced myeloid maturation of HL-60 cells : role of the transcription elongation block in the C-MYC gene

Proliferation-differentiation coupling was studied during dimethyl sulfoxide (DMSO)-induced myeloid maturation of HL-60 cells using transcription of the myeloperoxidase (MPO) and c-myc genes as indicators of differentiation and proliferation, respectively. Concomitant cell cycle kinetic analysis correlated the proliferation and transcription patterns. Transcription, cell cycle phases and rate of DNA synthesis were examined for up to 5 days of induction and, at 1-day intervals, analyzed during a 24-h reculture without the inducer. DMSO suppressed transcription of the c-myc and MPO genes with a t1/2 of 16 min and 7 h, respectively. The ability to recover transcription following reculture diminished with the progression of the induction and ultimately was lost; concomitantly, the cells irreversibly lost the capacity to divide. This indicated that the differentiation and proliferation processes are inseparable and that terminal differentiation accompanies irreversible proliferation arrest in HL-60 cells. We also studied the kinetics of the block to transcription elongation at the exon 1-intron 1 boundary of the c-myc gene. This block produces a 0.38 kb truncated transcript that is constitutively expressed in somatic cells (Re et al., Oncogene 5, 1247, 1990). During induction the level of the 0.38 kb RNA increased, while that of the complete c-myc mRNA decreased, indicating that this truncated RNA is generated instead of message through a monotonously initiated transcriptional process. Transcription initiation and synthesis of the 0.3 kb RNA persisted in terminally differentiated cells, suggesting a role for this RNA in non-proliferating cells.

Phosphorylation of Glutathione S-Transferase P1 (GSTP1) by Epidermal Growth Factor Receptor (EGFR) Promotes Formation of the GSTP1-c-Jun N-terminal kinase (JNK) Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells

Background: GSTP1 is a downstream EGFR phosphorylation target. Results: EGFR-dependent C-terminal Tyr-198 phosphorylation shifts GSTP1 to the monomeric state, facilitates JNK binding and inhibition, and suppresses apoptosis in brain tumor cells. Conclusion: Enhanced suppression of JNK signaling by EGFR-phosphorylated GSTP1 provides a survival advantage for tumors. Significance: The GSTP1-EGFR cross-talk is a mechanism of tumor cell survival and drug resistance. Under normal physiologic conditions, the glutathione S-transferase P1 (GSTP1) protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, GSTP1 binds to the mitogen-activated protein kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by epidermal growth factor receptor (EGFR) at tyrosine residues 3, 7, and 198. Here we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1, and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr-198 by EGFR and is associated with a >2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 cross-talk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs.

8;20 Chromosomal translocation in a case of acute leukemia: Cytogenetic, immunophenotypic, ultrastructural, and molecular characteristics

An 8;20 chromosomal translocation was observed in the leukemia cells of a 3-year-old girl. To our knowledge, this is the first report of this translocation in de novo acute leukemia. This chromosomal defect was present in the leukemia cells at diagnosis and also at relapse, but remission bone marrow cells had the 46,XX karyotype. By morphologic and cytochemical criteria the leukemia was myeloid but these features were more lymphoid when the leukemia recurred. However, the immunophenotype was consistent with myeloid leukemia and did not change at relapse. No evidence for either immunoglobulin or TCR gene rearrangement was observed.

Rational Design and Development of Targeted Brain Tumor Therapeutics

The majority of agents currently used to treat patients with tumors of the central nervous system (CNS) are cytotoxins, developed initially for their ability to kill cells directly. Subsequent drugs were derived by an analog strategy in which relatively small chemical modifications were made to the parent drug to either increase its potency or decrease its systemic toxicity, often with little to no change in the drug’s mechanism of action. This approach has yielded chemotherapeutic agents, notably DNA damaging agents, such as 2-chloroethylnitrosoureas (CENUs), nitrogen mustard-type drugs, and the platinum analogs that have been the mainstay of brain tumor chemotherapy to date.

Abstract 783: Mutational status of the p53 tumor suppressor gene is associated with expression of GSTP1 allelic variants in human GBM..

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The tumor suppressor and transcription factor, p53, regulates the transcription of a large number of genes that control key cellular functional, such as, cell cycle arrest, DNA repair, senescence and apoptosis, and its mutational inactivation of p53 has been implicated in tumor development and progression. Mutation in p53 is a common genetic abnormality in GBM and other cancers and several studies have associated wild type p53 with high tumor drug resistance. Our laboratory previously reported that the polymorphic human GSTP1 gene is a transcriptional target of and is activated by p53 via a canonical p53 binding motif located in intron 4 of the GSTP1 gene. The ability of the p53 wild type to transcriptionally activate the GSTP1 gene, thus, defines a novel mechanism of protecting the genome and enhancement tumor drug resistance. The ability of the different GSTP1 alleles to protect against the toxicity of anticancer agents plays an important factor in this GSTP1-mediated tumor drug resistance. In a previous study, we showed that p53 mutations in GBM spanned the entire p53 gene, with the majority of the mutations in the DNA binding domain and that GSTP1 expression correlated, in most cases with wild type p53. In the present study, we examined the relationship between p53-dependent GSTP1 gene activation genetic polymorphism and expression in human GBM. We sequenced the p53 gene in 52 primary human GBMs, genotyped these tumors for their GSTP1 allelic status and determined the expression of the GSTP1 gene transcripts by quantitative PCR. The results showed the GSTP1*A allele to most highly (40%) expressed in the tumors and of these 27% harbored wild-type p53. In contrast, GSTP1*C allele was not only the least prevalent allele but also had the least association with wild-type p53. GSTP1*B, as well as, heterozygous GSTP1 alleles were intermediate in their association with p53 mutational status of the tumors. The level of expression of the individual genes also correlated with the presence of wild-type p53 in inversely with mutant p53. These results demonstrate a significant impact of p53 mutational status, not only in mediating the level of GSTP1 gene expression but also that it is related to the specific allelic form of GSTP1 that is expressed. This suggest that p53 mutational status and both GSTP1 expression and allele polymorphism may interact together to mediate glioma drug resistance. Citation Format: Gamil R. Antoun, Francis Ali-Osman. Mutational status of the p53 tumor suppressor gene is associated with expression of GSTP1 allelic variants in human GBM. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 783. doi:10.1158/1538-7445.AM2013-783

Further Evidence that the Human Differentiation Antigen p24 Possesses Activity Associated with Protein Kinase

A fundamental premise underlying the study of differentiation is that the individual molecules on the surface of a cell, at a particular stage of maturation, have a functional role. This role may involve either the main-tenance of the individual cell or the regulation of its interaction with other cells and possibly the microenvironment. Significant clarification of the nature of the molecular composition of the cell surface during commitment to lineage and differentiation has been provided by investigations utilizing murine monoclonal antibodies (mAbs). The determination of the functional role played by these molecules has recently been addressed.

Abstract 1177: The mutational status of the p53 tumor suppressor gene is a determinant of GSTP1 expression and mediates GSTP1-dependent drug resistance in human glioblastoma

Mutations in the p53 tumor suppressor gene, a transcription factor activated by a variety of cellular stresses, including, those inflicted by anti-cancer agents, is a common genetic abnormality in GBMs. Although, p53 is involved in critical cellular processes, such as, cell cycle arrest, DNA repair and apoptosis, studies of p53 in tumor drug resistance and/or patient response to therapy have yielded mixed results. Thus, in gliomas and other cancers, such as, breast, bladder, and ovarian carcinomas, some studies have associated wild-type p53 with higher tumor drug resistance, while other studies indicate that mutant or functionally inactive p53, rather than wt p53, confers drug resistance. These observations indicate the significant complexity of the role of p53 in tumor response to therapy and suggest that other factors, including, downstream p53 pathways and/or regulation, may be important determinants of the p53 effects on the tumor drug resistance phenotype. We previously reported that the human GSTP1 gene, that encodes a major drug metabolizing protein, cell signaling regulator and a mediator of tumor drug resistance, is transcriptionally activated by p53 via a canonical p53 binding motif located in intron 4 of the GSTP1 gene. Our goal, in this study, is to gain further insight into the relationship between p53-dependent transcriptional control of GSTP1 in GBM and the extent to which it contributes to GSTP1 expression in primary patient tumor and, ultimately, to their response to therapy. For this, we sequenced the p53 gene across exons 2-11 in 42 primary GBM specimens quantified the level of GSTP1 gene expression by RNA-PCR and genotyped the allelic variants in them. The results showed, approx. half of the tumors to harbor wild-type p53. In those with mutant p53, the mutations spanned the entire p53 target region, the majority in the core DNA binding domain with the hot spots in codons 55, 72, 175, 245, 248, and 273. GSTP1 expression was significantly heterogeneous between the tumors, and with a few exceptions, correlated directly with the presence of wild-type p53. In representative tumors, the high GSTP1 gene expression was associated with resistance to cisplatin and temozolomide. These result suggest that, in GBM, transcriptional activation of GSTP1 by wild-type, and some mutant forms of p53, contributes to their resistance to therapy. Supported by NIH grants RO1 CA 153050, RO1CA127872, RO1 CA 112519, P50CA108786 and P30-CA14236. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1177. doi:1538-7445.AM2012-1177