Manjula Agarwal

Involvement of human MOF in ATM function

ABSTRACT We have determined that hMOF, the human ortholog of the Drosophila MOF gene (males absent on the first), encoding a protein with histone acetyltransferase activity, interacts with the ATM (ataxia-telangiectasia-mutated) protein. Cellular exposure to ionizing radiation (IR) enhances hMOF-dependent acetylation of its target substrate, lysine 16 (K16) of histone H4 independently of ATM function. Blocking the IR-induced increase in acetylation of histone H4 at K16, either by the expression of a dominant negative mutant ΔhMOF or by RNA interference-mediated hMOF knockdown, resulted in decreased ATM autophosphorylation, ATM kinase activity, and the phosphorylation of downstream effectors of ATM and DNA repair while increasing cell killing. In addition, decreased hMOF activity was associated with loss of the cell cycle checkpoint response to DNA double-strand breaks. The overexpression of wild-type hMOF yielded the opposite results, i.e., a modest increase in cell survival and enhanced DNA repair after IR exposure. These results suggest that hMOF influences the function of ATM.

The mammalian ortholog of Drosophila MOF that acetylates histone H4 lysine 16 is essential for embryogenesis and oncogenesis.

ABSTRACT The mammalian ortholog of the Drosophila MOF (males absent on the first) gene product is a histone H4 lysine 16-specific acetyltransferase. Recent studies have shown that depletion of human MOF (hMOF) in human cell lines leads to genomic instability, spontaneous chromosomal aberrations, cell cycle defects, altered nuclear morphology, reduced transcription of certain genes, and defective DNA damage response to ionizing radiation (IR). Here we show that MOF plays an essential role in mammals during embryogenesis and oncogenesis. Ablation of the mouse Mof gene (mMof) by gene targeting resulted in early embryonic lethality and cell death. Lethality correlated with the loss of H4 lysine 16 acetylation (H4K16ac) and could not be rescued by concomitant inactivation of ATM or p53. In comparison to primary cells or normal tissue, all immortalized human normal and tumor cell lines and primary tumors demonstrated similar or elevated hMOF and H4K16ac levels. Accordingly, MOF overexpression correlated with increased cellular proliferation, oncogenic transformation, and tumor growth. Thus, these data reveal that the acetylation of histone H4 at K16 by MOF is an epigenetic signature of cellular proliferation common to both embryogenesis and oncogenesis and that MOF is an essential factor for embryogenesis and oncogenesis.

Hyperthermia Activates a Subset of Ataxia-Telangiectasia Mutated Effectors Independent of DNA Strand Breaks and Heat Shock Protein 70 Status

All cells have intricately coupled sensing and signaling mechanisms that regulate the cellular outcome following exposure to genotoxic agents such as ionizing radiation (IR). In the IR-induced signaling pathway, specific protein events, such as ataxia-telangiectasia mutated protein (ATM) activation and histone H2AX phosphorylation (gamma-H2AX), are mechanistically well characterized. How these mechanisms can be altered, especially by clinically relevant agents, is not clear. Here we show that hyperthermia, an effective radiosensitizer, can induce several steps associated with IR signaling in cells. Hyperthermia induces gamma-H2AX foci formation similar to foci formed in response to IR exposure, and heat-induced gamma-H2AX foci formation is dependent on ATM but independent of heat shock protein 70 expression. Hyperthermia also enhanced ATM kinase activity and increased cellular ATM autophosphorylation. The hyperthermia-induced increase in ATM phosphorylation was independent of Mre11 function. Similar to IR, hyperthermia also induced MDC1 foci formation; however, it did not induce all of the characteristic signals associated with irradiation because formation of 53BP1 and SMC1 foci was not observed in heated cells but occurred in irradiated cells. Additionally, induction of chromosomal DNA strand breaks was observed in IR-exposed but not in heated cells. These results indicate that hyperthermia activates signaling pathways that overlap with those activated by IR-induced DNA damage. Moreover, prior activation of ATM or other components of the IR-induced signaling pathway by heat may interfere with the normal IR-induced signaling required for chromosomal DNA double-strand break repair, thus resulting in increased cellular radiosensitivity.

Human Heterochromatin Protein 1 Isoforms HP1Hsα and HP1Hsβ Interfere with hTERT-Telomere Interactions and Correlate with Changes in Cell Growth and Response to Ionizing Radiation

ABSTRACT Telomeres are associated with the nuclear matrix and are thought to be heterochromatic. We show here that in human cells the overexpression of green fluorescent protein-tagged heterochromatin protein 1 (GFP-HP1) or nontagged HP1 isoforms HP1Hsα or HP1Hsβ, but not HP1Hsγ, results in decreased association of a catalytic unit of telomerase (hTERT) with telomeres. However, reduction of the G overhangs and overall telomere sizes was found in cells overexpressing any of these three proteins. Cells overexpressing HP1Hsα or HP1Hsβ also display a higher frequency of chromosome end-to-end associations and spontaneous chromosomal damage than the parental cells. None of these effects were observed in cells expressing mutants of GFP-ΔHP1Hsα, GFP-ΔHP1Hsβ, or GFP-ΔHP1Hsγ that had their chromodomains deleted. An increase in the cell population doubling time and higher sensitivity to cell killing by ionizing radiation (IR) treatment was also observed for cells overexpressing HP1Hsα or HP1Hsβ. In contrast, cells expressing mutant GFP-ΔHP1Hsα or GFP-ΔHP1Hsβ showed a decrease in population doubling time and decreased sensitivity to IR compared to the parental cells. The effects on cell doubling times were paralleled by effects on tumorigenicity in mice: overexpression of HP1Hsα or HP1Hsβ suppressed tumorigenicity, whereas expression of mutant HP1Hsα or HP1Hsβ did not. Collectively, the results show that human cells are exquisitely sensitive to the amount of HP1Hsα or HP1Hsβ present, as their overexpression influences telomere stability, population doubling time, radioresistance, and tumorigenicity in a mouse xenograft model. In addition, the isoform-specific effects on telomeres reinforce the notion that telomeres are in a heterochromatinized state.

Role for proteasome activator PA200 and postglutamyl proteasome activity in genomic stability

Proteasome activator PA200 enhances proteasome-mediated cleavage after acidic residues in vitro; however, its role within cells is not known. Here, we show that, in response to ionizing radiation, PA200 forms hybrid proteasomes with 19S caps and 20S core proteasomes that accumulate on chromatin, leading to an increase in proteolytic activity. Unlike many other proteins that respond to DNA damage, the response of PA200 appears to be independent of Ataxia Telangiectasia Mutated and p53, but dependent on DNA-dependent protein kinase activity. Nonetheless, PA200 is critical because PA200-knockdown cells show genomic instability and reduced survival after exposure to ionizing radiation. This phenotype is reproduced by specific inhibition of postglutamyl activity of proteasomes, but combined treatment with PA200 siRNA and postglutamyl inhibitor does not show additive effects on survival. Together, these data suggest a unique role for PA200 in genomic stability that is likely mediated through its ability to enhance postglutamyl cleavage by proteasomes.

Inhibition of Telomerase Activity Enhances Hyperthermia- Mediated Radiosensitization

Hyperthermia is a potent sensitizer of cell killing by ionizing radiation (IR); however, hyperthermia also induces heat shock protein 70 (HSP70) synthesis and HSP70 expression is associated with radioresistance. Because HSP70 interacts with the telomerase complex and expression of the telomerase catalytic unit (hTERT) extends the life span of the human cells, we determined if heat shock influences telomerase activity and whether telomerase inhibition enhances heat-mediated IR-induced cell killing. In the present study, we show that moderate hyperthermia (43 degrees C) enhances telomerase activity. Inhibition of telomerase activity with human telomerase RNA-targeted antisense agents, and in particular GRN163L, results in enhanced hyperthermia-mediated IR-induced cell killing, and ectopic expression of catalytic unit of telomerase (TERT) decreased hyperthermia-mediated IR-induced cell killing. The increased cell killing by heat and IR exposure in telomerase-inhibited cells correlates with delayed appearance and disappearance of gamma-H2AX foci as well as decreased chromosome repair. These results suggest that inactivation of telomerase before combined hyperthermia and radiotherapy could improve tumor killing.

Inhibitors of HGFA, Matriptase, and Hepsin Serine Proteases: A Nonkinase Strategy to Block Cell Signaling in Cancer

Hepatocyte growth factor activators (HGFA), matriptase, and hepsin are S1 family trypsin-like serine proteases. These proteases proteolytically cleave the single-chain zymogen precursors, pro-HGF (hepatocyte growth factor), and pro-MSP (macrophage stimulating protein) into active heterodimeric forms. HGF and MSP are activating ligands for the oncogenic receptor tyrosine kinases (RTKs), c-MET and RON, respectively. We have discovered the first substrate-based ketothiazole inhibitors of HGFA, matriptase and hepsin. The compounds were synthesized using a combination of solution and solid-phase peptide synthesis (SPPS). Compounds were tested for protease inhibition using a kinetic enzyme assay employing fluorogenic peptide substrates. Highlighted HGFA inhibitors are Ac-KRLR-kt (5g), Ac-SKFR-kt (6c), and Ac-SWLR-kt (6g) with K is = 12, 57, and 63 nM, respectively. We demonstrated that inhibitors block the conversion of native pro-HGF and pro-MSP by HGFA with equivalent potency. Finally, we show that inhibition causes a dose-dependent decrease of c-MET signaling in MDA-MB-231 breast cancer cells. This preliminary investigation provides evidence that HGFA is a promising therapeutic target in breast cancer and other tumor types driven by c-MET and RON.

Abstract LB-197: Hepatocyte Growth Factor Activator (HGFA) Inhibitors of c-MET/RON Kinase Signaling

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Continuous advances have been made in the treatment of cancer however 70% of the patients still die of cancer every year strongly correlated to tumor metastasis. The treatment options available at present are rarely able to effectively treat metastatic cancer. Targeting hepatocyte growth factor (HGF) signaling with receptor tyrosine kinase (RTK) inhibitors is a well-developed strategy for metastatic cancer therapy however no studies have explored targets upstream of kinase activation. One such target is hepatocyte growth factor activator (HGFA) which is upregulated in a large number of cancer cell lines and patient samples. HGFA is a trypsin-like serine protease which stimulates both the c-MET and RON signaling pathways by proteolysis of the inactive single-chain zymogen growth factor ligand precursors to activated two-chain HGF and macrophage stimulating protein (MSP) respectively. Increased activation of these pathways has been shown to trigger a number of downstream signaling events necessary for metastasis including cell migration, invasion, proliferation, and angiogenesis. The present study was undertaken to discover inhibitors of HGFA based on the naturally occurring bicyclic peptide, sunflower trypsin inhibitor (SFTI). Using a combination of solution and solid-phase peptide methods, we have synthesized the natural product SFTI and analogs thereof. The compounds were tested for HGFA activity in a competitive fluorescence resonance energy transfer (FRET) assay of proteolytic cleavage by using a fluorogenic peptide substrate. We have identified several inhibitors of HGFA with low μM activity in the FRET assay. We have also demonstrated one inhibitor effectively prevented the conversion of native pro-HGF to HGF as determined by western blotting in a biochemical assay of HGFA activity. This ground breaking discovery of novel HGFA inhibitors will enable future studies on the role of HGFA and other serine proteases in the regulation of growth factors, cell signaling, and cancer metastasis. Inhibitors of HGFA offer an innovative therapeutic strategy upstream of kinase activation and signaling for the treatment of metastatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-197. doi:10.1158/1538-7445.AM2011-LB-197