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Supplementary table

Supplementary table

Supplementary Figure

Figure S4. Sensitivity to AZD1775 in ATRX WT and KO PLC/PRF/5 cells after adjusting for differences in growth rates.

Supplementary table

Purpose:Metallothionein 2A (MT2A) suppresses the progression of human gastric cancer potentially through an “MT2A–NF-κB pathway” with unclear mechanisms. This study explored the role of a transcription factor, myeloid zinc-finger 1 (MZF1), in MT2A-NF-κB pathway and its clinical significance in gastric cancer.Experimental Design:MZF1 expression and function in gastric cancer were investigated in vitro and in vivo. The relationship between MZF1 and MT2A was determined by gain-of-function and loss-of-function assays in gastric cancer cells and an immortalized gastric cell line GES-1. The prognostic value of MZF1 expression in association with MT2A was evaluated using IHC in two cohorts.Results:MZF1 was epigenetically silenced in human gastric cancer cell lines and primary tumors. Overexpression of MZF1 in gastric cancer cells suppressed cell proliferation and migration, as well as the growth of xenograft tumors in nude mice. Knocking-down of MZF1 transformed GES-1 cells into a malignant phenotype characterized by increased cell growth and migration. Mechanistically, MZF1 was upregulated in both GC and GES-1 cells by MT2A ectopically expressed or induced upon treatment with a garlic-derived compound, diallyl trisulfide (DATS). MZF1 associated with MT2A was colocalized in the nuclei of GES-1 cells to target the promoter of NF-κB inhibitor alpha (NFKBIA). Clinically, MT2A and MZF1 were progressively downregulated in clinical specimens undergoing gastric malignant transformation. Downregulation of MT2A and MZF1 was significantly correlated with poorer patient prognosis.Conclusions:MT2A exerts its anti-gastric cancer effects by complexing with MZF1 to target NFKBIA. MT2A/MZF1 may serve as a valuable prognostic marker and a novel therapeutic target for human gastric cancer.

Supplementary Materials and Methods

Figure S2. MAGeCK mle beta scores for all genes in ATRX WT and ATRX KO PLC/PRF/5 cells.

Supplementary Figure

Supplementary Figure

The growth rates of ATRX mutant and wildtype glioma cells treated by AZD1775.

Supplementary table

The tumor suppressor gene ATRX is frequently mutated in a variety of tumors including gliomas and liver cancers, which are highly unresponsive to current therapies. Here, we performed a genome-wide synthetic lethal screen, using CRISPR-Cas9 genome editing, to identify potential therapeutic targets specific for ATRX-mutated cancers. In isogenic hepatocellular carcinoma (HCC) cell lines engineered for ATRX loss, we identified 58 genes, including the checkpoint kinase WEE1, uniquely required for the cell growth of ATRX null cells. Treatment with the WEE1 inhibitor AZD1775 robustly inhibited the growth of several ATRX-deficient HCC cell lines in vitro, as well as xenografts in vivo. The increased sensitivity to the WEE1 inhibitor was caused by accumulated DNA damage–induced apoptosis. AZD1775 also selectively inhibited the proliferation of patient-derived primary cell lines from gliomas with naturally occurring ATRX mutations, indicating that the synthetic lethal relationship between WEE1 and ATRX could be exploited in a broader spectrum of human tumors. As WEE1 inhibitors have been investigated in several phase II clinical trials, our discovery provides the basis for an easily clinically testable therapeutic strategy specific for cancers deficient in ATRX.Significance:ATRX-mutant cancer cells depend on WEE1, which provides a basis for therapeutically targeting WEE1 in ATRX-deficient cancers.See related commentary by Cole, p. 375

Figure S1. Validation of ATRX KO in PLC/PRF/5 and HuH-7 cells.

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Supplementary Figure S3. Validation of ATRX synthetic lethal candidates by RNAi.

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Supplementary table

Western blots displaying the effects of ATRX knockdown in PLC/PRF/5 and HeLa cells.

Treatment of AZD1775 activated CDK pathway, which leads to S-phase arrest and DNA damage, and finally apoptosis of cells.

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Supplementary Figure

The tumor suppressor gene ATRX is frequently mutated in a variety of tumors including gliomas and liver cancers, which are highly unresponsive to current therapies. Here, we performed a genome-wide synthetic lethal screen, using CRISPR-Cas9 genome editing, to identify potential therapeutic targets specific for ATRX-mutated cancers. In isogenic hepatocellular carcinoma (HCC) cell lines engineered for ATRX loss, we identified 58 genes, including the checkpoint kinase WEE1, uniquely required for the cell growth of ATRX null cells. Treatment with the WEE1 inhibitor AZD1775 robustly inhibited the growth of several ATRX-deficient HCC cell lines in vitro, as well as xenografts in vivo. The increased sensitivity to the WEE1 inhibitor was caused by accumulated DNA damage–induced apoptosis. AZD1775 also selectively inhibited the proliferation of patient-derived primary cell lines from gliomas with naturally occurring ATRX mutations, indicating that the synthetic lethal relationship between WEE1 and ATRX could be exploited in a broader spectrum of human tumors. As WEE1 inhibitors have been investigated in several phase II clinical trials, our discovery provides the basis for an easily clinically testable therapeutic strategy specific for cancers deficient in ATRX. Significance: ATRX-mutant cancer cells depend on WEE1, which provides a basis for therapeutically targeting WEE1 in ATRX-deficient cancers. See related commentary by Cole, p. 375

Loss of chromosome 7 and del(7q) [-7/del(7q)] are recurring cytogenetic abnormalities in acute myeloid leukemia (AML), occurring in 9% of de novo AML and 50% of therapy-related myeloid neoplasms (t-MN). Abnormalities of chromosome 7 carry an adverse prognosis. Despite intensive analysis by many laboratories, and the identification of multiple minimally deleted regions on 7q, the putative tumor suppressor(s) on chromosome 7 is currently unknown. We performed transcriptome sequencing on 22 samples and SNP array analysis on 35 samples from patients with myeloid neoplasms, half of which contain -7/del(7q). Copy number analysis identified a 2.17 Mb commonly deleted segment on chromosome band 7q22.1 containing CUX1, a gene encoding a homeodomain-containing transcription factor normally expressed highly in hematopoietic stem cells. CUX1 was found to be disrupted in a translocation resulting in a chimeric RNA fusion transcript in one patient. We found the CUX1 transcript and protein to be expressed at haploinsufficent levels in -7/del(7q) leukemias. To test the tumor suppressor activity of CUX1, the drosophila homologue, CUT, was knocked-down in Drosophila melanogaster hemocytes, which led to significantly increased numbers of hemocytes and melanotic tumor formation. Restoration of CUX1 expression in a human AML cell line with -7 decreased proliferation. These data indicate that CUX1 is a conserved, haploinsufficient, tumor suppressor that promotes leukemogenesis in -7/del(7q) AML. 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 LB-411. doi:1538-7445.AM2012-LB-411