Donald Chow

Translational Genomics Research Institute, Phoenix, Arizona, United States

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Publications (8)75.78 Total impact

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    ABSTRACT: Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified anti-apoptotic BCL-2 family members as potent 5-Azacytidine sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared to the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. Based on these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.Leukemia accepted article preview online, 23 January 2014. doi:10.1038/leu.2014.44.
    Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 01/2014; · 10.16 Impact Factor
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    ABSTRACT: Deregulation of the TWEAK-Fn14 signaling pathway is observed in many diseases including inflammation, autoimmune diseases, and cancer. Activation of Fn14 signaling by TWEAK binding triggers cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK [Tyrosine 176 (Y176) and Tryptophan 231 (W231)], were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Y176, but not W231, resulted in the loss of TWEAK binding to Fn14 substantiating Y176 as the anchoring residue. Importantly, mutation of TWEAK at Y176 did not disrupt TWEAK trimerization, but failed to induce Fn14 mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results validate the TWEAK-Fn14 interaction as a chemically tractable target and provide the foundation for further exploration utilizing chemical biology approaches focusing on validating this system as a therapeutic target in invasive cancers.
    Journal of Biological Chemistry 09/2013; · 4.65 Impact Factor
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    ABSTRACT: Synucleinopathies are a broad class of neurodegenerative disorders characterized by the presence of intracellular protein aggregates containing α-synuclein protein. The aggregated α-synuclein protein is hyperphosphorylated on serine 129 (S129) compared to the unaggregated form of the protein. While the precise functional consequences of S129 hyperphosphorylation are still being clarified, numerous in vitro and in vivo studies suggest that S129 phosphorylation is an early event in α-synuclein dysfunction and aggregation. Identifying the kinases and phosphatases that regulate this critical phosphorylation event may ultimately prove beneficial by allowing pharmacological mitigation of synuclein dysfunction and toxicity in Parkinson's disease and other synucleinopathies. We report here the development of a high-content, fluorescence-based assay to quantitate levels of total and S129 phosphorylated α-synuclein protein. We have applied this assay to conduct high-throughput loss-of-function screens with siRNA libraries targeting 711 known and predicted human kinases and 206 phosphatases. Specifically, knockdown of the phosphatidylinositol 3-kinase related kinase SMG1 resulted in significant increases in the expression of pS129 phosphorylated α-synuclein (p-syn). Moreover, SMG1 protein levels were significantly reduced in brain regions with high p-syn levels in both dementia with Lewy bodies (DLB) and Parkinson's disease with dementia (PDD). These findings suggest that SMG1 may play an important role in increased α-synuclein pathology during the course of PDD, DLB, and possibly other synucleinopathies.
    PLoS ONE 01/2013; 8(10):e77711. · 3.73 Impact Factor
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    ABSTRACT: To identify rational therapeutic combinations with cytarabine (Ara-C), we developed a high-throughput, small-interference RNA (siRNA) platform for myeloid leukemia cells. Of 572 kinases individually silenced in combination with Ara-C, silencing of 10 (1.7%) and 8 (1.4%) kinases strongly increased Ara-C activity in TF-1 and THP-1 cells, respectively. The strongest molecular concepts emerged around kinases involved in cell-cycle checkpoints and DNA-damage repair. In confirmatory siRNA assays, inhibition of WEE1 resulted in more potent and universal sensitization across myeloid cell lines than siRNA inhibition of PKMYT1, CHEK1, or ATR. Treatment of 8 acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) cell lines with commercial and the first-in-class clinical WEE1 kinase inhibitor MK1775 confirmed sensitization to Ara-C up to 97-fold. Ex vivo, adding MK1775 substantially reduced viability in 13 of 14 AML, CML, and myelodysplastic syndrome patient samples compared with Ara-C alone. Maximum sensitization occurred at lower to moderate concentrations of both drugs. Induction of apoptosis was increased using a combination of Ara-C and MK1775 compared with using either drug alone. WEE1 is expressed in primary AML, ALL, and CML specimens. Data from this first siRNA-kinome sensitizer screen suggests that inhibiting WEE1 in combination with Ara-C is a rational combination for the treatment of myeloid and lymphoid leukemias.
    Blood 01/2012; 119(12):2863-72. · 9.06 Impact Factor
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    ABSTRACT: We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.
    Nature Genetics 12/2011; 44(2):165-9. · 35.21 Impact Factor
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    ABSTRACT: YB-1 is a multifunctional protein that affects transcription, splicing, and translation. Overexpression of YB-1 in breast cancers causes cisplatin resistance. Recent data have shown that YB-1 is also overexpress in colorectal cancer. In this study, we tested the hypothesis that YB-1 also confers oxaliplatin resistance in colorectal adenocarcinomas. We show for the first time that transfection of YB-1 cDNA confers oxaliplatin resistance in two colorectal cancer cell lines (SW480 and HT29 cell lines). Furthermore, we identified by mass spectrometry analyses important YB-1 interactors required for such oxaliplatin resistance in these colorectal cancer cell lines. A tagged YB-1 construct was used to identify proteins interacting directly to YB-1 in such cells. We then focused on proteins that are potentially involved in colorectal cancer progression based on the Oncomine microarray database. Genes encoding for these YB-1 interactors were also examined in the public NCBI comparative genomic hybridization database to determine whether these genes are localized to regions of chromosomes rearranged in colorectal cancer tissues. From these analyses, we obtained a list of proteins interacting with YB-1 and potentially involved in oxaliplatin resistance. Oxaliplatin dose response curves of SW480 and HT29 colorectal cancer cell lines transfected with several siRNAs corresponding to each of these YB-1 interactors were obtained to identify proteins significantly affecting oxaliplatin sensitivity upon gene silencing. Only the depletion of either NONO or RALY sensitized both colorectal cancer cell lines to oxaliplatin. Furthermore, depletion of NONO or RALY sensitized otherwise oxaliplatin resistant overexpressing YB-1 SW480 or HT29 cells. These results suggest knocking down NONO or RALY significant counteracts oxaliplatin resistance in colorectal cancers overexpressing the YB-1 protein.
    Molecular Cancer 11/2011; 10:145. · 5.13 Impact Factor
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    ABSTRACT: The Y-box binding protein 1 (YB-1) is a multifunctional protein that affects transcription, splicing, and translation. Overexpression of YB-1 in breast cancers causes cisplatin resistance. The exact mechanism by which YB-1 confers cisplatin resistance is unknown. The aim of the present study was to identify, using mass spectrometry, proteins that interact with YB-1 that are important for cisplatin resistance in two breast cancer cell lines, namely MCF7 and MDA-MB-231. A tagged YB-1 construct was used to identify proteins interacting directly with YB-1 in breast cancer cells. We then focused on proteins that are potentially involved in breast cancer progression based on the ONCOMINE public microarray database. Genes encoding for these YB-1-interacting proteins were examined in the public NCBI comparative genomic hybridization database to determine whether they are localized to regions of chromosomes that are rearranged in breast cancer tissues. From these analyses, we generated a list of proteins potentially involved in cisplatin resistance. Cisplatin dose–response curves were constructed in MCF7 and MDA-MB-231 transfected with four siRNA corresponding to each of these YB-1 interactors to identify proteins significantly affecting cisplatin sensitivity upon gene silencing. Depletion of only the X-linked ribosomal protein S4 (RPS4X) resulted in consistent resistance to cisplatin in both cell lines with at least three different siRNA sequences against RPS4X. Further analyses indicated that the knock down of RPS4X decreased DNA synthesis, induced cisplatin resistance, and is equivalent to the overexpression of YB-1 in both MCF7 and MDA-MB-231 cells. These results suggest that the RPS4X/YB-1 complex is a significant potential target to counteract cisplatin resistance in breast cancer. (Cancer Sci 2011; 102: 1410–1417)
    Cancer Science 05/2011; 102(7):1410 - 1417. · 3.48 Impact Factor
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    ABSTRACT: Oxaliplatin is widely used to treat colorectal cancer, as both adjuvant therapy for resected disease and palliative treatment of metastatic disease. However, a significant number of patients experience serious side effects, including prolonged neurotoxicity, from oxaliplatin treatment creating an urgent need for biomarkers of oxaliplatin response or resistance to direct therapy to those most likely to benefit. As a first step to improve selection of patients for oxaliplatin-based chemotherapy, we have conducted an in vitro cell-based small interfering RNA (siRNA) screen of 500 genes aimed at identifying genes whose loss of expression alters tumor cell response to oxaliplatin. The siRNA screen identified twenty-seven genes, which when silenced, significantly altered colon tumor cell line sensitivity to oxaliplatin. Silencing of a group of putative resistance genes increased the extent of oxaliplatin-mediated DNA damage and inhibited cell-cycle progression in oxaliplatin-treated cells. The activity of several signaling nodes, including AKT1 and MEK1, was also altered. We used cDNA transfection to overexpress two genes (LTBR and TMEM30A) that were identified in the siRNA screen as mediators of oxaliplatin sensitivity. In both instances, overexpression conferred resistance to oxaliplatin. In summary, this study identified numerous putative predictive biomarkers of response to oxaliplatin that should be studied further in patient specimens for potential clinical application. Diverse gene networks seem to influence tumor survival in response to DNA damage by oxaliplatin. Finally, those genes whose loss of expression (or function) is related to oxaliplatin sensitivity may be promising therapeutic targets to increase patient response to oxaliplatin.
    Molecular Cancer Research 02/2011; 9(2):173-82. · 4.35 Impact Factor