Manali Phadke

Chromatin-associated proteins HMGB1/2 and PDIA3 trigger cellular response to chemotherapy-induced DNA damage

The identification of new molecular components of the DNA damage signaling cascade opens novel avenues to enhance the efficacy of chemotherapeutic drugs. High-mobility group protein 1 (HMGB1) is a DNA damage sensor responsive to the incorporation of nonnatural nucleosides into DNA; several nuclear and cytosolic proteins are functionally integrated with HMGB1 in the context of DNA damage response. The functional role of HMGB1 and HMGB1-associated proteins (high-mobility group protein B2, HMGB2; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; protein disulfide isomerase family A member 3, PDIA3; and heat shock 70 kDa protein 8, HSPA8) in DNA damage response was assessed in human carcinoma cells A549 and UO31 by transient knockdown with short interfering RNAs. Using the cell proliferation assay, we found that knockdown of HMGB1-associated proteins resulted in 8-fold to 50-fold decreased chemosensitivity of A549 cells to cytarabine. Western blot analysis and immunofluorescent microscopy were used to evaluate genotoxic stress markers in knocked-down cancer cells after 24 to 72 hours of incubation with 1 μmol/L of cytarabine. Our results dissect the roles of HMGB1-associated proteins in DNA damage response: HMGB1 and HMGB2 facilitate p53 phosphorylation after exposure to genotoxic stress, and PDIA3 has been found essential for H2AX phosphorylation (no γ-H2AX accumulated after 24–72 hours of incubation with 1 μmol/L of cytarabine in PDIA3 knockdown cells). We conclude that phosphorylation of p53 and phosphorylation of H2AX occur in two distinct branches of the DNA damage response. These findings identify new molecular components of the DNA damage signaling cascade and provide novel promising targets for chemotherapeutic intervention.[Mol Cancer Ther 2009;8(4):864–72]

Glyceraldehyde 3-Phosphate Dehydrogenase Depletion Induces Cell Cycle Arrest and Resistance to Antimetabolites in Human Carcinoma Cell Lines

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that acts at the intersection of energy metabolism and stress response in tumor cells. To elucidate the role of GAPDH in chemotherapy-induced stress, we analyzed its activity, protein level, intracellular distribution, and intranuclear mobility in human carcinoma cells A549 and UO31 after treatment with cytarabine, doxorubicin, and mercaptopurine. After treatment with cytosine arabinoside (araC), enzymatically inactive GAPDH accumulated in the nucleus. Experiments on fluorescence recovery after photobleaching with green fluorescent protein-GAPDH fusion protein in the live cells treated with araC demonstrated reduced mobility of green fluorescent protein-GAPDH inside the nucleus, indicative of interactions with nuclear macromolecular components after genotoxic stress. Depletion of GAPDH with RNA interference stopped cell proliferation, and induced cell cycle arrest in G1 phase via p53 stabilization, and accumulation of p53-inducible CDK inhibitor p21. Neither p21 accumulation nor cell cycle arrest was detected in GAPDH-depleted p53-null NCI-H358 cells. GAPDH-depleted A549 cells were 50-fold more resistant to treatment with cytarabine (1.68 ± 0.182 μM versus 0.03 ± 0.015 μM in control). Depletion of GAPDH did not significantly alter cellular sensitivity to doxorubicin (0.05 ± 0.023 μM versus 0.035 ± 0.0154 μM in control). Induction of cell cycle arrest in p53-proficient carcinoma cells via GAPDH abrogation suggests that GAPDH-depleting agents may have a cytostatic effect in cancer cells. Our results define GAPDH as an important determinant of cellular sensitivity to antimetabolite chemotherapy because of its regulatory functions.

Synthesis and Evaluation of Strychnos Alkaloids as MDR Reversal Agents for Cancer Cell Eradication

Natural products represent the fourth generation of multidrug resistance (MDR) reversal agents that resensitize MDR cancer cells overexpressing P-glycoprotein (Pgp) to cytotoxic agents. We have developed an effective synthetic route to prepare various Strychnos alkaloids and their derivatives. Molecular modeling of these alkaloids docked to a homology model of Pgp was employed to optimize ligand-protein interactions and design analogues with increased affinity to Pgp. Moreover, the compounds were evaluated for their (1) binding affinity to Pgp by fluorescence quenching, and (2) MDR reversal activity using a panel of in vitro and cell-based assays and compared to verapamil, a known inhibitor of Pgp activity. Compound 7 revealed the highest affinity to Pgp of all Strychnos congeners (Kd=4.4μM), the strongest inhibition of Pgp ATPase activity, and the strongest MDR reversal effect in two Pgp-expressing cell lines. Altogether, our findings suggest the clinical potential of these synthesized compounds as viable Pgp modulators justifies further investigation.

Accelerated cellular senescence phenotype of GAPDH-depleted human lung carcinoma cells

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a pivotal glycolytic enzyme, and a signaling molecule which acts at the interface between stress factors and the cellular apoptotic machinery. Earlier, we found that knockdown of GAPDH in human carcinoma cell lines resulted in cell proliferation arrest and chemoresistance to S phase-specific cytotoxic agents. To elucidate the mechanism by which GAPDH depletion arrests cell proliferation, we examined the effect of GAPDH knockdown on human carcinoma cells A549. Our results show that GAPDH-depleted cells establish senescence phenotype, as revealed by proliferation arrest, changes in morphology, SA-β-galactosidase staining, and more than 2-fold up-regulation of senescence-associated genes DEC1 and GLB1. Accelerated senescence following GAPDH depletion results from compromised glycolysis and energy crisis leading to the sustained AMPK activation via phosphorylation of α subunit at Thr172. Our findings demonstrate that GAPDH depletion switches human tumor cells to senescent phenotype via AMPK network, in the absence of DNA damage. Rescue experiments using metabolic and genetic models confirmed that GAPDH has important regulatory functions linking the energy metabolism and the cell cycle networks. Induction of senescence in LKB1-deficient non-small cell lung cancer cells via GAPDH depletion suggests a novel strategy to control tumor cell proliferation.

Cytotoxicity of chemotherapeutic agents in glyceraldehyde-3-phosphate dehydrogenase-depleted human lung carcinoma A549 cells with the accelerated senescence phenotype.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays a central role in glycolysis. Because cancer cells rely on aerobic glycolysis rather than oxidative phosphorylation, GAPDH-depleting agents have a therapeutic potential to impede cancer cell proliferation. Knockdown of GAPDH by RNA interference induced the accelerated senescent phenotype in A549 cells, suggesting that GAPDH is a potential molecular target for combination chemotherapy. The cytotoxic effects of a panel of anticancer drugs, 5-fluorouracil, 5-fluorouridine, 5-fluorodeoxyuridine, 6-thioguanine, cytarabine, fludarabine, cladribine, clofarabine, 2-chloroadenosine, and doxorubicin, were assessed in GAPDH-depleted A549 cells using a cell proliferation assay. GAPDH-depleted A549 cells, when compared with control cells, exhibited increased chemoresistance to several antimetabolite agents including cytarabine [inhibitory concentration 50 (IC50) 1.7±0.3 vs. 0.03±0.02 &mgr;mol/l], 2-chloroadenosine (IC50 7.1±1.8 vs. 1.5±0.6 &mgr;mol/l), 6-thioguanine (IC50 7.5±1.6 vs. 1.4±0.5 &mgr;mol/l), 5-fluorouracil (IC50 13.2±2.5 vs. 3.0±0.7 &mgr;mol/l), and 5-fluorodeoxyuridine (IC50 >100 vs. 3.7±0.9 &mgr;mol/l), which we designated as group A agents. In contrast, GAPDH-deficient and GAPDH-proficient cells were equally sensitive to group B agents including doxorubicin (IC50 0.05±0.02 vs. 0.04±0.02 &mgr;mol/l), fludarabine (IC50 18.5±2.3 vs. 15.7±2.8 &mgr;mol/l), 5-fluorouridine (IC50 0.1±0.03 vs. 0.1±0.03 &mgr;mol/l), clofarabine (IC50 0.7±0.3 vs. 0.5±0.3 &mgr;mol/l), and cladribine (IC50 0.5±0.1 vs. 0.5±0.2 &mgr;mol/l). After treatment with group B agents at concentrations equivalent to 7–10-fold the IC50 value, the fraction of apoptotic cells in GAPDH-depleted, senescent A549 cells was similar to that in GAPDH-proficient cells. Our study identified the antimetabolite drugs active in senescent cells that can be used in combination with GAPDH inhibitors in cancer treatment. GAPDH-targeted combination therapy is a novel strategy to control the proliferation of tumor cells.

The novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 overcomes acquired BRAF inhibitor resistance through suppression of Mcl-1 and MEK expression

Resistance to BRAF inhibitors is a major clinical problem. Here, we evaluate BI-847325, an ATP-competitive inhibitor of MEK and Aurora kinases, in treatment-naïve and drug-resistant BRAF-mutant melanoma models. BI-847325 potently inhibited growth and survival of melanoma cell lines that were both BRAF inhibitor naïve and resistant in 2D culture, 3D cell culture conditions, and in colony formation assays. Western blot studies showed BI-847325 to reduce expression of phospho-ERK and phospho-histone 3 in multiple models of vemurafenib resistance. Mechanistically, BI-847325 decreased the expression of MEK and Mcl-1 while increasing the expression of the proapoptotic protein BIM. Strong suppression of MEK expression was observed after 48 hours of treatment, with no recovery following >72 hours of washout. siRNA-mediated knockdown of Mcl-1 enhanced the effects of BI-847325, whereas Mcl-1 overexpression reversed this in both 2D cell culture and 3D spheroid melanoma models. In vivo, once weekly BI-847325 (70 mg/kg) led to durable regression of BRAF-inhibitor naïve xenografts with no regrowth seen (>65 days of treatment). In contrast, treatment with the vemurafenib analog PLX4720 was associated with tumor relapse at >30 days. BI-847325 also suppressed the long-term growth of xenografts with acquired PLX4720 resistance. Analysis of tumor samples revealed BI-847325 to induce apoptosis associated with suppression of phospho-ERK, total MEK, phospho-Histone3, and Mcl-1 expression. Our studies indicate that BI-847325 is effective in overcoming BRAF inhibitor resistance and has long-term inhibitory effects upon BRAF-mutant melanoma in vivo, through a mechanism associated with the decreased expression of both MEK and Mcl-1. Mol Cancer Ther; 14(6); 1354–64. ©2015 AACR.

Dabrafenib inhibits the growth of BRAF‐WT cancers through CDK16 and NEK9 inhibition

Although the BRAF inhibitors dabrafenib and vemurafenib have both proven successful against BRAF‐mutant melanoma, there seem to be differences in their mechanisms of action. Here, we show that dabrafenib is more effective at inhibiting the growth of NRAS‐mutant and KRAS‐mutant cancer cell lines than vemurafenib. Using mass spectrometry‐based chemical proteomics, we identified NEK9 and CDK16 as unique targets of dabrafenib. Both NEK9 and CDK16 were highly expressed in specimens of advanced melanoma, with high expression of both proteins correlating with a worse overall survival. A role for NEK9 in the growth of NRAS‐ and KRAS‐mutant cell lines was suggested by siRNA studies in which silencing was associated with decreased proliferation, cell cycle arrest associated with increased p21 expression, inhibition of phospho‐CHK1, decreased CDK4 expression, and the initiation of a senescence response. Inhibition of CDK4 but not CHK1 recapitulated the effects of NEK9 silencing, indicating this to be the likely mechanism of growth inhibition. We next turned our attention to CDK16 and found that its knockdown inhibited the phosphorylation of the Rb protein at S780 and increased expression of p27. Both of these effects were phenocopied in NRAS‐ and KRAS‐mutant cancer cells by dabrafenib, but not vemurafenib. Combined silencing of NEK9 and CDK16 was associated with enhanced inhibition of melanoma cell proliferation. In summary, we have identified dabrafenib as a potent inhibitor of NEK9 and CDK16, and our studies suggest that inhibition of these kinases may have activity against cancers that do not harbor BRAF mutations.

Abstract 703: A novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 reverses acquired BRAF inhibitor resistance through suppression of Mcl-1 and inhibition of MEK expression

BACKGROUND: BRAF/MEK inhibitors have shown promising levels of response in melanomas harboring the BRAF V600E mutation, however responses tend to be short-lived and resistance is a major clinical problem. In the current study, we evaluated the pharmacological activity of BI847325, a dual inhibitor of MEK and Aurora kinases against multiple melanoma cell line models. METHODS: The cytotoxic effect of BI847325 was evaluated in vitro in a panel of selected BRAF-mutant vemurafenib resistant cell lines by Alamar blue and Annexin V binding assay. 3D spheroid model systems and colony formation assays demonstrated the long-term growth inhibitory effect of BI847325. Western blot analysis and qRT-PCR studies were carried out to evaluate the mechanism underlying BI5-mediated cytotoxicity. In vivo studies in Balb SCID mice were performed to assess the suppression of BRAF-mutant xenografts on treatment with BI847325. RESULTS: BI847325 potently reduced the growth and survival of BRAF-mutant melanoma cell lines with acquired and intrinsic BRAF inhibitor resistance (NRAS mutations, BRAF splice forms, Cyclin D1 amplification, RTK upregulation, PTEN loss, COT amplification). We confirmed that BI847325 induced apoptosis through decrease in Mcl-1 mRNA and protein expression and increase in BIM expression. The effects of BI847325 upon BIM and Mcl-1 expression could not be mimicked by the combination of other MEK and aurora kinase inhibitors. For the first time we demonstrated that BI847325 reverses the acquired vemurafenib resistance by inhibiting MEK expression at mRNA and protein level. A strong suppression of MEK expression was observed without recovery following 72 h of washout. In vivo studies revealed complete tumor suppression with no recurrence over a period of 65 days of treatment with 70mg/kg/week dose of BI847325. In contrast, treatment with vemurafenib analog PLX4720 in the same mouse model was associated with tumor relapse after 30 days of treatment. BI847325 also successfully suppressed the long-term growth of xenografts with acquired vemurafenib resistance. Analysis of tumor samples complied with in vitro results demonstrating inhibition of phospho-ERK, phospho-Histone3, Mcl-1 and total MEK. CONCLUSION: In conclusion we report for the first time that BI847325, a novel ATP-competitive MEK/ Aurora kinase inhibitor effectively inhibits BRAF-mutant melanoma and overcomes vemurafenib-resistance by decreasing expression of MEK and Mcl-1; in vitro and in vivo. Further preclinical and clinical investigations towards this would open new avenues in treatment of melanoma. Citation Format: Manali S. Phadke, Patrizia Sini, Keiran Smalley. A novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 reverses acquired BRAF inhibitor resistance through suppression of Mcl-1 and inhibition of MEK expression. [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 703. doi:10.1158/1538-7445.AM2015-703

Abstract 1579: Depletion of GAPDH promotes p53 signaling, cell senescence and altered response to chemotherapeutic agents in human carcinoma cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a pivotal enzyme of the glycolytic pathway; it also plays a role of a signaling/regulator molecule in several cellular networks. In this study, we used human lung carcinoma A549 and renal carcinoma UO-31 cell lines to elucidate the role of GAPDH in cell proliferation and cellular response to genotoxic drugs. Using RNA interference technology, we decreased GAPDH protein level 4-5-fold in A549 cells, and 2-3-fold in UO-31 cells. Depletion of GAPDH in cultured cells blocked cell cycle progression in G1 phase, and induced activation of p53 and accumulation of p21. Simultaneously, GAPDH-depleted cells manifested biomarkers of senescence as revealed by morphological changes and induction of SA-β-galactosidase activity. Inhibition of GAPDH synthesis in human carcinoma cells resulted in 3-4-fold ATP depletion, and induced cell growth arrest without AMPK phosphorylation. In contrast, inhibition of glycolysis by 2-deoxyglucose treatment resulted in decreased ATP level and AMPK phosphorylation but did not induce cell senescence. These results suggest that GAPDH depletion in A549 cell line activates p53 independently of AMPK activation, and mediates the induction of signaling pathways leading to senescence. Importantly, GAPDH knockdown in p53-proficient A549 and UO-31 cells resulted in 3-50-fold decreased cytotoxicity of antimetabolite drugs including deoxy-thioguanosine, cladribine, fluoro-ribouridine, fluoruracil, 2-chloroadenosine, mercaptopurine, azacytidine and cytarabine. The results of this study indicate that cytotoxic activity of antimetabolite nucleoside analogs is modulated by the status of GAPDH in the cancer cells. Cell senescence induced by depletion of GAPDH is a plausible mechanism of cell resistance to antimetabolites, a hypothesis being under investigation in our lab. In addition, our data indicate that GAPDH-depleting agents could be promising cytostatic drugs. This work was supported by NCI grant R01 CA104729 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1579.