MAGE proteins regulate KRAB zinc finger transcription factors and KAP1 E3 ligase activity.

Expression of Melanoma AntiGen Encoding (MAGE) genes, particularly MAGE-A3, has been correlated with aggressive clinical course, the acquisition of resistance to chemotherapy and poor clinical outcomes of melanoma and other malignancies. MAGE proteins bind to KAP1, a gene repressor and ubiquitin E3 ligase which also binds KRAB domain containing zinc finger transcription factors (KZNFs), and MAGE expression may affect KZNF mediated gene regulation. To investigate mechanisms for these effects, we tested the hypothesis that differences in KRAB domain composition affect KZNF poly-ubiquitination and determine whether MAGE expression increases, decreases, or has no effect on KZNFs mediated gene repression. Using an integrated reporter gene responsive to repression by KRAB domain fusion proteins, we found that MAGE-A3 relieved KZNF mediated repression and induced KZNF poly-ubiquitination and degradation in association with expression of the A+B box KRAB domain. In contrast, MAGE-A3 enhanced KAP1 mediated repression of KZNFs expressing A or A+b box KRAB domains but caused no increase in poly-ubiquitination or degradation. MAGE-A3 has no significant impact on KZNFs with KRAB domains containing the Scan box motif. These data support our hypothesis by showing that the effects of MAGE-A3 on gene repression depend on the type of KZNF KRAB domain involved.

MAGE-C2 promotes growth and tumorigenicity of melanoma cells, phosphorylation of KAP1, and DNA damage repair.

Melanoma-associated antigen-encoding (MAGE) genes are expressed in melanoma and other cancers but not in normal somatic cells. MAGE expression is associated with aggressive tumor growth, poor clinical outcome, and resistance to chemotherapy, but the mechanisms have not been completely elucidated. In this study, we show that downregulation of MAGE-C2 in A375 melanoma cells and low-passage cultures from human metastatic melanomas (MRA cells) results in increased apoptosis and decreased growth of tumor xenografts in athymic nude mice. Previously, we showed that MAGE-C2 binds KAP1, a scaffolding protein that regulates DNA repair. Phosphorylation of KAP1-Serine 824 (Ser824) by ataxia-telangiectasia-mutated (ATM) kinase is necessary for repair of DNA double-strand breaks (DSBs); now we show that MAGE-C2 knockdown reduces, whereas MAGE-C2 overexpression increases, ATM kinase-dependent phosphorylation of KAP1-Ser824. We demonstrate that MAGE-C2 increases co-precipitation of KAP1 with ATM and that binding of MAGE-C2 to KAP1 is necessary for increased KAP1-Ser824 phosphorylation. Furthermore, ectopic expression of MAGE-C2 enhances repair of I-SceI endonuclease-induced DSBs in U-2OS cells. As phosphorylation of KAP1-Ser824 facilitates relaxation of heterochromatin, which is necessary for DNA repair and cellular proliferation, our results suggest that MAGE-C2 can promote tumor growth by phosphorylation of KAP1-Ser824 and by enhancement of DNA damage repair.

T cell and B Cell immunity can be reconstituted with mismatched hematopoietic stem cell transplantation without alkylator therapy in artemis-deficient mice using anti-natural killer cell antibody and photochemically treated sensitized donor T cells.

Children with Artemis-deficient T(-)B(-)NK(+) severe combined immunodeficiency are at high risk for graft rejection from natural killer (NK) cells and toxicity from increased sensitivity to the alkylating agents used in mismatched hematopoietic stem cell transplantation (HSCT). We evaluated the use of a nonalkylating agent regimen before HSCT in Artemis-deficient (mArt(-/-)) C57Bl/6 (B6) mice to open marrow niches and achieve long-term multilineage engraftment with full T cell and B cell immune reconstitution. We found that partial depletion of both recipient NK cells using anti-NK1.1 monoclonal antibody and donor T cells sensitized to recipient splenocytes was necessary. BALB/c-sensitized T cells (STCs) were photochemically treated (PCT) with psoralen and UVA light to inhibit proliferation, reduce the risk of graft-versus-host disease (GVHD), and target host hematopoietic stem cells (HSCs). A dose of 4 × 10(5) PCT STCs coinjected with 1 × 10(5) lineage-depleted c-kit(+) BALB/c HSCs resulted in 43.9% ± 3.3% CD4(+) and 10.9% ± 1.2% CD8(+) donor T cells in blood, 29% ± 7.8% and 21.7% ± 4.0 donor B220(+) IgM(+) in spleen and bone marrow, and 15.0% ± 3.6% donor Gran-1(+) cells in bone marrow at 6 months post-HSCT versus 0.02% ± 0.01%, 0.13% ± 0.10%, 0.53% ± 0.16%, 0.49% ± 0.09%, and 0.20% ± 0.06%, respectively, in controls who did not receive PCT STCs. We found that STCs target host HSCs and that PCT STCs are detectable only up to 24 hours after infusion, in contrast to non-photochemically treated STCs, which proliferate resulting in fatal GVHD. Increased mortality in the groups receiving 4-6 × 10(5) PCT STCs was associated with evidence of GVHD, particularly in the recipients of 6 × 10(5) cells. These results demonstrate that blocking NK cell-mediated resistance and making niches in bone marrow are both essential to achieving multilineage engraftment of mismatched donor cells and T cell and B cell reconstitution, even though GVHD is not completely eliminated.

MAGE I transcription factors regulate KAP1 and KRAB domain zinc finger transcription factor mediated gene repression.

Class I MAGE proteins (MAGE I) are normally expressed only in developing germ cells but are aberrantly expressed in many cancers. They have been shown to promote tumor survival, aggressive growth, and chemoresistance but the underlying mechanisms and MAGE I functions have not been fully elucidated. KRAB domain zinc finger transcription factors (KZNFs) are the largest group of vertebrate transcription factors and regulate neoplastic transformation, tumor suppression, cellular proliferation, and apoptosis. KZNFs bind the KAP1 protein and direct KAP1 to specific DNA sequences where it suppresses gene expression by inducing localized heterochromatin characterized by histone 3 lysine 9 trimethylation (H3me3K9). Discovery that MAGE I proteins also bind to KAP1 prompted us to investigate whether MAGE I can affect KZNF and KAP1 mediated gene regulation. We found that expression of MAGE I proteins, MAGE-A3 or MAGE-C2, relieved repression of a reporter gene by ZNF382, a KZNF with tumor suppressor activity. ChIP of MAGE I (-) HEK293T cells showed KAP1 and H3me3K9 are normally bound to the ID1 gene, a target of ZNF382, but that binding is greatly reduced in the presence of MAGE I proteins. MAGE I expression relieved KAP1 mediated ID1 repression, causing increased expression of ID1 mRNA and ID1 chromatin relaxation characterized by loss of H3me3K9. MAGE I binding to KAP1 also induced ZNF382 poly-ubiquitination and degradation, consistent with loss of ZNF382 leading to decreased KAP1 binding to ID1. In contrast, MAGE I expression caused increased KAP1 binding to Ki67, another KAP1 target gene, with increased H3me3K9 and decreased Ki67 mRNA expression. Since KZNFs are required to direct KAP1 to specific genes, these results show that MAGE I proteins can differentially regulate members of the KZNF family and KAP1 mediated gene repression.

Identification of novel small molecules that inhibit protein-protein interactions between MAGE and KAP-1.

The Class I MAGE proteins are normally expressed only in developing germ cells but are often aberrantly expressed in malignancies, particularly melanoma, making them good therapeutic targets. MAGE proteins promote tumor survival by binding to the RBCC region of KAP-1 and suppressing p53. Although, suppression of MAGE expression, by RNA interference, relieves p53 suppression and inhibits tumor growth, its therapeutic uses are limited by lack of methods for systemic delivery of small interfering RNA. To overcome this barrier, we sought to discover chemical compounds that inhibit binding between MAGE and KAP-1 proteins. Based on previously published effects of MAGE suppression, we developed a strategy for screening a small molecule library based on selective death of MAGE positive cells, activation of p53 and lack of caspase activity. We screened the Maybridge HitFinder library of compounds and eight compounds fulfilled these criteria. Seven of these compounds interfered with co-precipitation of MAGE and KAP-1, and three interfered with binding of MAGE and KAP-1 in a mammalian two hybrid assay. We now report identification of three potential compounds that interfere with MAGE/KAP-1 binding and can be developed as novel chemo-therapeutic agents for treatment of advanced melanoma and other cancers.