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In vivo phosphoproteome

Supplementary Figures and Methods

Fanconi anemia (FA) is a genome instability syndrome that has been associated with both cancer predisposition and bone marrow failure. FA proteins are involved in cellular response to replication stress in which they coordinate DNA repair with DNA replication and cell-cycle progression. One regulator of the replication stress response is the ATP-dependent DNA translocase FANCM, which we have shown to be hyperphosphorylated in response to various genotoxic agents. However, the significance of this phosphorylation remained unclear. Here, we show that genotoxic stress–induced FANCM phosphorylation is ATR-dependent and that this modification is highly significant for the cellular response to replication stress. We identified serine (S1045) residue of FANCM that is phosphorylated in response to genotoxic stress and this effect is ATR-dependent. We show that S1045 is required for FANCM functions including its role in FA pathway integrity, recruiting FANCM to the site of interstrand cross links, preventing the cells from entering mitosis prematurely, and efficient activation of the CHK1 and G2–M checkpoints. Overall, our data suggest that an ATR-FANCM feedback loop is present in the FA and replication stress response pathways and that it is required for both efficient ATR/CHK1 checkpoint activation and FANCM function. Cancer Res; 73(14); 4300–10. ©2013 AACR.

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PDF file - 407K, DNA-damage-induced phosphorylation of FANCM is ATR dependent (S1); Peptides released from FANCM protein were analyzed for putative phosphorylation sites by mass spectrometry (S2); Immunoblot showing that antibody raised against phosphorylated S1045 specifically recognizes the ectopically expressed wildtype, but not S1045A mutant, form of FANCM (S3); UV mediated DNA-damage-induced phosphorylation of FANCM at S1045 (S4); Phosphorylation at S1045 is not required for FANCM binding to the FA core complex but for FANCD2 monoubiquitination (S5); MMC-induced chromatin association of the FA-core complex is impaired in S1045A-expressing cells (S6); Recruitment of FANCM to ICL site is ATR dependent (S7); S1045A mutant-expressing cells do not show any gross change in cell-cycle distribution (S8); Expression of FLAG tagged FANCMK117R in the context of knockdown of the endogenous protein (S9).

Purpose: Somatic inactivating mutations in ARID1A, a component of the SWI/SNF chromatin remodeling complex, are detected in various types of human malignancies. Loss of ARID1A compromises DNA damage repair. The induced DNA damage burden may increase reliance on PARP-dependent DNA repair of cancer cells to maintain genome integrity and render susceptibility to PARP inhibitor therapy. Experimental Design: Isogenic ARID1A−/− and wild-type cell lines were used for assessing DNA damage response, DNA compactness, and profiling global serine/threonine phosphoproteomic in vivo. A panel of inhibitors targeting DNA repair pathways was screened for a synergistic antitumor effect with irradiation in ARID1A−/− tumors. Results: ARID1A-deficient endometrial cells exhibit sustained levels in DNA damage response, a result further supported by in vivo phosphoproteomic analysis. Our results show that ARID1A is essential for establishing an open chromatin state upon DNA damage, a process required for recruitment of 53BP1 and RIF1, key mediators of non-homologous end-joining (NHEJ) machinery, to DNA lesions. The inability of ARID1A−/− cells to mount NHEJ repair results in a partial cytotoxic response to radiation. Small-molecule compound screens revealed that PARP inhibitors act synergistically with radiation to potentiate cytotoxicity in ARID1A−/− cells. Combination treatment with low-dose radiation and olaparib greatly improved antitumor efficacy, resulting in long-term remission in mice bearing ARID1A-deficient tumors. Conclusions: ARID1A-deficient cells acquire high sensitivity to PARP inhibition after exposure to exogenously induced DNA breaks such as ionizing radiation. Our findings suggest a novel biologically informed strategy for treating ARID1A-deficient malignancies.

Resveratrol (trans-3,4′,5-trihydroxystilbene) is a naturally occurring polyphenolic compound highly enriched in grapes, peanuts, red wine, and a variety of food sources. Resveratrol has antiinflammatory and antioxidant properties, and also has potent anticancer properties. Human glioma U251 cells were used to understand the molecular mechanisms by which resveratrol acts as an anticancer agent, since glioma is a particularly difficult cancer to treat and eradicate. Our data show that resveratrol induces dose- and time-dependent death of U251 cells, as measured by lactate dehydrogenase release and internucleosomal DNA fragmentation assays. Resveratrol induces activation of caspase-3 and increases the cleavage of the downstream caspase substrate, poly(ADP-ribose) polymerase. Resveratrol-induced DNA fragmentation can be completely blocked by either a general caspase inhibitor (Z-VAD-FMK) or a selective caspase-3 inhibitor (Z-DEVD-FMK), but not by a selective caspase-1 inhibitor. Resveratrol induces cytochrome c release from mitochondria to the cytoplasm and activation of caspase-9. Resveratrol also increases expression of proapoptotic Bax and its translocation to the mitochondria. Resveratrol inhibits U251 proliferation, as measured by MTS assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], and induces G0/G1 growth arrest, as determined by flow cytometry. The cyclin-dependent kinase inhibitor, olomoucine, prevents cell cycle progression and resveratrol-induced apoptosis. These results suggest that multiple signaling pathways may underlie the apoptotic death of U251 glioma induced by resveratrol, which warrants further exploration as an anticancer agent in human glioma.

Poly (ADP-ribose) polymerase (PARP) inhibitors represent one of the most exciting recent developments in cancer therapy. While substantial efficacy has been shown with clinically available PARP inhibitors (PARPis), to date, in treatment of hereditary deletions of BRCA1/2 in breast and ovarian cancers, the high promise of these drugs has not yet been realized in sporadic cancers. We present here strong preclinical data for a novel, mechanistically based, combinatorial approach to using DNA methyltransferase inhibitors (DNMTis), such as decitabine (DAC) and 5-Azacytidine (5-AZA), with PARP inhibitors (PARPis) as a treatment strategy for acute myelogenous leukemias (AML) and triple negative breast cancer (TNBC). We have previously demonstrated that low doses of 5-AZA and DAC alone show efficacy in AML and TNBC, and propose treatment with PARPis to enhance sensitivity of cancer cells to DNMTis. The mechanistic rationale for our approach is based upon: 1) data from our group and others showing DNMT1 and PARP1 associate in a complex, and this association increases with DNA damage; 2) the fact that 5-AZA and DAC trap DNMTs led us to hypothesize that these drugs might also increase PARP trapping at DNA damage sites; and 3) the cytotoxicity of the most potent PARPis (e.g. BMN 673) appears to correlate with the degree of trapping of PARP1 in chromatin. We first find that in cultured human AML and TNBC cells, the DNMTis (5 to 20 nM DAC or 100 to 200nM 5-AZA) and PARPis (1 to 10 nM BMN 673) alone trap PARP into chromatin, and this effect is enhanced when the drugs are combined. In addition, the PARPi-DNMTi combination treatment of TNBC cell line MDA-MB-231 resulted in significantly enhanced retention of PARP1 and DNMT1 at sites of double strand breaks (DSBs) induced by laser microirradiation. Concomitant with this, the combined doses resulted in significant increases in cytotoxic DSBs, observed 4-24 hours after DSB induction, when compared to single-drug treatments. Homologous recombination (HR) DSB repair activity also appears decreased, as measured by GFP reporter assays. In keeping with these findings, colony survival assays demonstrated that the combination treatment, compared to either drug alone, strongly inhibited colony formation of TNBC cell lines (N=4). Notably non-tumorigenic MCF10A cells showed no significant differences in colony numbers with single or combination drug treatments. Similar to TNBCs, AML cell lines (N=3) as well as primary AML cells (N=8) showed dramatic decreases in colonies in combination vs single agent drug treatments. In the most important translational implications of the preliminary studies, in in vivo therapy TNBC and AML models in immune-deficient mice, our low dose combinations of DNMTis and PARPis provide for potent anti-tumor responses. Mouse xenograft experiments using BRCA mutant TNBC cell line SUM149PT demonstrated that the combination treatment has a significant (p Citation Format: Nidal Muvarak, Khadiza Chowdhury, Carine Robert, Xia Limin, Eun Yong Choi, Yi Cai, Marina Bellani, Michael Seidman, Maria R. Baer, Rena Lapidus, Stephen B. Baylin, Feyruz V. Rassool. Combination of DNA methyltransferase and PARP inhibitors as a novel therapy strategy for multiple cancers: Key data in AML and triple negative breast cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA13.

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) have shown efficacy in treatment of breast and ovarian cancers with hereditary deletions of BRCA1/2, but the high promise of these drugs has not yet been realized in sporadic cancers. We present here strong preclinical data for a novel, mechanistically based, combinatorial approach to using DNA methyltransferase inhibitors (DNMTi’s), such as decitabine (DAC) and 5-Azacytidine (5-AZA), with PARP inhibitors (PARPi’s) as a treatment strategy for acute myelogenous leukemias (AML) and estogen-, progesterone- and HER2-receptor negative, or triple negative breast cancer (TNBC). We have previously demonstrated that low doses of 5-AZA and DAC alone show efficacy in AML and TNBC, and propose treatment with PARPi's to enhance sensitivity of cancer cells to DNMTis. The mechanistic rationale for our approach is based upon: 1) data from our group and others showing DNMT1 and PARP1 associate in a complex, and this association increases with DNA damage; 2) the fact that 5-AZA and DAC trap DNMT's led us to hypothesize that these drugs might also increase PARP trapping at DNA damage sites; and 3) the cytotoxicity of the most potent PARPi's (e.g. Talazoparib) appears to correlate with the degree of trapping of PARP1 in chromatin. We find that in cultured human AML and TNBC cells, the DNMTi's (5 to 20 nM DAC or 100 to 200nM 5-AZA) and PARPi's (1 to 10 nM Talazoparib) alone trap PARP into chromatin, and this effect is enhanced when the drugs are combined. In addition, the PARPi-DNMTi combination treatment in TNBC MDA-MB-231 and AML MOLM-14 cell lines resulted in significantly increased DNA double strand breaks (DSBs) and enhanced retention of PARP1 and DNMT1 at laser microirradiation DNA damage sites. Compared with non-tumorigenic MCF10A cells, in TNBC cell lines (N = 4), the combined doses resulted in significant (p Citation Format: Feyruz V. Rassool, Nidal Muvarak, Khadiza Chowdury, Carine Robert, Limin Xia, Eun Yong Choi, Yi Cai, Marina Bellani, Ying Zou, Michael Seidman, Søren Bentzen, Maria Baer, Rena Lapidus, Stephen B. Baylin. Combination of DNA methyltransferase and PARP inhibitors as a novel therapy strategy for poor prognosis acute myeloid leukemia and triple-negative breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-205.

Regulation of dormant origin firing under conditions of replication stress is poorly understood. The Bloom syndrome DNA helicase BLM functions in maintaining replication fork stability, with sumoylation regulating this function, and BLM deficiency is associated with increased dormant origin firing. We found here that BLM sumoylation levels increase in response to replication stalling by hydroxyurea (HU) and to proteasome inhibition; depletion of the SUMO-targeted ubiquitin ligase RNF4 also causes increased levels of BLM sumoylation. RNF4 directly interacts with BLM and can ubiquitylate sumoylated BLM in vitro. These data indicate that sumoylated BLM is an RNF4 substrate. RNF4 is recruited to DNA repair foci in response to replication stalling. RNF4 depletion causes the accumulation of increased numbers of HU-induced BLM foci, whereas the numbers of RPA, RAD51, and gamma-H2AX foci are unaffected by fork stalling in RNF4-depleted cells. Sister chromatid exchanges are normal in RNF4-depleted cells. These data indicate that RNF4 can regulate BLM retention at stalled forks without affecting repair by homologous recombination. RNF4 depletion reduces cell proliferation and survival of untreated cells, and it confers hypersensitivity to replication stalling by HU. Studies of cell-cycle progression using bromodeoxyuridine incorporation and flow cytometry show that RNF4 depletion causes a delay of re-entry into S phase in HU-treated cells and the replication delay is partially rescued by BLM mutation. The delay is not caused by modification of checkpoint kinase activities. Analysis of replication dynamics using the DNA fiber assay show that RNF4 depletion causes an increase of permanently stalled replication forks and a decrease in activation of dormant origins following recovery from HU-induced replication stress. Co-depletion of RNF4 and BLM partially rescues these defects. Replication dynamics is unaffected by RNF4 depletion in untreated cells. These data indicate that RNF4 regulates BLM's functions in replication fork stability and dormant origin firing, without affecting BLM's roles in homologous recombination. We propose that sumoylation of BLM at stalled replication forks leads to RNF4-mediated ubiquitylation and subsequent proteasome-dependent degradation of BLM, which relieves BLM's active inhibition of dormant origin firing. Citation Format: Nathan A. Ellis, Wei-Chih Yang, Mary Yagle, Jianmei Zhu, Jing Huang, Michael Seidman, Michael J. Matunis. The SUMO-targeted ubiquitin ligase RNF4 regulates BLM helicase's function in dormant origin firing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3036. doi:10.1158/1538-7445.AM2015-3036