• Targeting RAD52 DNA binding domain I by peptide aptamer induces synthetic lethality in BRCA-deficient leukemias.• Individual patients with BRCA-deficient leukemias could be identified by genetic and epigenetic profiling.Homologous recombination repair (HRR) protects cells from the lethal effect of spontaneous and therapy-induced DNA double-stand breaks. HRR usually depends on BRCA1/2-RAD51, and RAD52-RAD51 serves as back-up. To target HRR in tumor cells, a phenomenon called "synthetic lethality" was applied, which relies on the addiction of cancer cells to a single DNA repair pathway, whereas normal cells operate 2 or more mechanisms. Using mutagenesis and a peptide aptamer approach, we pinpointed phenylalanine 79 in RAD52 DNA binding domain I (RAD52-phenylalanine 79 [F79]) as a valid target to induce synthetic lethality in BRCA1-and/or BRCA2-deficient leukemias and carcinomas without affecting normal cells and tissues. Targeting RAD52-F79 disrupts the RAD52-DNA interaction, resulting in the accumulation of toxic DNA double-stand breaks in malignant cells, but not in normal counterparts. In addition, abrogation of RAD52-DNA interaction enhanced the antileukemia effect of already-approved drugs. BRCA-deficient status predisposing to RAD52-dependent synthetic lethality could be predicted by genetic abnormalities such as oncogenes BCR-ABL1 and PML-RAR, mutations in BRCA1 and/or BRCA2 genes, and gene expression profiles identifying leukemias displaying low levels of BRCA1 and/or BRCA2. We believe this work may initiate a personalized therapeutic approach in numerous patients with tumors displaying encoded and functional BRCA deficiency. (Blood. 2013;122(7):1293-1304

Cell cycle progression is intricately controlled by coordinated expression and ubiquitin-dependent degradation of key regulatory proteins. Ubiquitylation of target substrates requires the concerted activity of an Ub-activating enzyme (E1), recruitment of an Ub-conjugating enzyme (E2), and an E3 ubiquitin ligase. Although some E3 ubiquitin ligases directly mediate transfer of ubiquitin, for example, the HECT-domain family of E3 ligases, many E3 ligases function as multi-subunit complexes that bridge the charged E2 enzyme with a given substrate (20). The Skp1-Cul1-F-box (SCF) family of E3 ubiquitin ligases is an example of the latter and promotes polyubiquitylation of mainly phosphorylated substrates, including G 1 /S regulatory proteins (11,12,27,32,43). Specificity of SCF ligases is conferred by the F-box protein, which bridges substrate molecules with the core ligase machinery. F-box proteins contain a conserved F-box domain, required for Skp1 binding and recruitment of core ligase components Cul1, Rbx1, and E2, as well as a substrate-recognition domain within the C terminus (39, 43, 51).The F-box protein, Fbx4, regulates ubiquitylation of cyclin D1, following the G 1 /S transition (32). Similar to Fbw7, a ubiquitin ligase for targets such as cyclin E, c-Myc, and Notch (27, 47-50, 54, 55), and ␤-TrCP, an E3 ligase for targets including IB, ␤-catenin, Cdc25A, and Emi1 (10,16,18,24,26,28,36,38,45,46,52), Fbx4 undergoes dimerization; however, in contrast to either Fbw7 or ␤-TrCP, Fbx4 dimerization occurs in a cell cycle-dependent manner (5,17,44,46). Fbx4 dimerization requires glycogen synthase kinase 3␤ (GSK3␤)-dependent phosphorylation at serine 12, which triggers ligase activation at the G 1 /S transition (5, 32). Critically, mutations that stabilize cyclin D1 and thereby trigger nuclear accumulation of active cyclin D1/CDK4 drive cell transformation (1-3). While cyclin D1 overexpression occurs in multiple human malignancies (4, 6-8, 14, 19, 21-23, 25), overexpression of wild-type cyclin D1 in an Fbx4-proficient system is insufficient to drive spontaneous transformation (3, 5), supporting a model wherein cytoplasmic recognition of phosphorylated cyclin D1 by Fbx4 is sufficient to maintain cellular integrity.Cyclin D1 mutations that impede degradation and trigger nuclear accumulation promote neoplastic growth (1,3,5,9,35,37); however, such mutations are rare in human cancer. Recent work identified inactivating Fbx4 mutations in human cancers that impair ligase phosphorylation and dimerization. Such tumors exhibit marked cyclin D1 nuclear accumulation, highlighting a novel mechanism for cyclin D1 deregulation in cancer (5). Taken together, these findings suggest that Fbx4 is a tumor suppressor, preventing aberrant cyclin D1 accumulation.To investigate the tumor suppressor function of Fbx4, we ablated the murine Fbx4 gene. Fbx4 Ϫ/Ϫ mice are viable and lack major developmental defects. However, Fbx4Ϫ/Ϫ murine embryonic fibroblasts (MEFs) exhibit cyclin D1 stabilization and subsequent nuclear localization, with con...

Key Points Normal ABL1 is a tumor suppressor in BCR-ABL1–induced leukemia. Allosteric stimulation of the normal ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors.

With this study we have demonstrated that in vitro transduction of normal human CD4(+) T lymphocytes with NPM-ALK results in their malignant transformation. The transformed cells become immortalized and display morphology and immunophenotype characteristic of patient-derived anaplastic large-cell lymphomas. These unique features, which are strictly dependent on NPM-ALK activity and expression, include perpetual cell growth, proliferation, and survival; activation of the key signal transduction pathways STAT3 and mTORC1; and expression of CD30 (the hallmark of anaplastic large-cell lymphoma) and of immunosuppressive cytokine IL-10 and cell-surface protein PD-L1/CD274. Implantation of NPM-ALK-transformed CD4(+) T lymphocytes into immunodeficient mice resulted in formation of tumors indistinguishable from patients' anaplastic large-cell lymphomas. Our findings demonstrate that the key aspects of human carcinogenesis closely recapitulating the features of the native tumors can be faithfully reproduced in vitro when an appropriate oncogene is used to transform its natural target cells; this in turn points to the fundamental role in malignant cell transformation of potent oncogenes expressed in the relevant target cells. Such transformed cells should permit study of the early stages of carcinogenesis, and in particular the initial oncogene-host cell interactions. This experimental design could also be useful for studies of the effects of early therapeutic intervention and likely also the mechanisms of malignant progression.

Here we report that cutaneous T-cell lymphoma (CTCL) cells and tissues ubiquitously express the immunosuppressive cell-surface protein CD80 (B7-1). CD80 expression in CTCL cells is strictly dependent on the expression of both members of STAT5 family: STAT5a and STAT5b and their joint ability to transcriptionally activate the CD80 gene. In the IL-2-dependent CTCL cells, CD80 expression is induced by the cytokine in the Jak1/3 and STAT5a/b-dependent manner, while in the CTCL cells with the constitutive STAT5 activation, CD80 expression is also STAT5a/b-dependent but independent of Jak activity. While depletion of CD80 expression does not affect the proliferative rate and viability of the CTCL cells, induced expression of the cell-inhibitory receptor of CD80; CD152 (CTLA-4), impairs growth of the cells. Co-culture of CTCL cells with normal T lymphocytes comprised of either both CD4+ and CD8+ populations, or the CD4+ subset alone, transfected with CD152 mRNA, inhibits proliferation of the normal T-cells in the CD152- and CD80-dependent manner. These data identify a new mechanism of immune evasion in CTCL and suggest that the CD80-CD152 axis may become a therapeutic target in this type of lymphoma.