Leronlimab, a humanized monoclonal antibody to CCR5, blocks breast cancer cellular metastasis and enhances cell death induced by DNA damaging chemotherapy.

BACKGROUND: Triple-negative breast cancer (BCa) (TNBC) is a deadly form of human BCa with limited treatment options and poor prognosis. In our prior analysis of over 2200 breast cancer samples, the G protein-coupled receptor CCR5 was expressed in > 95% of TNBC samples. A humanized monoclonal antibody to CCR5 (leronlimab), used in the treatment of HIV-infected patients, has shown minimal side effects in large patient populations. METHODS: A humanized monoclonal antibody to CCR5, leronlimab, was used for the first time in tissue culture and in mice to determine binding characteristics to human breast cancer cells, intracellular signaling, and impact on (i) metastasis prevention and (ii) impact on established metastasis. RESULTS: Herein, leronlimab was shown to bind CCR5 in multiple breast cancer cell lines. Binding of leronlimab to CCR5 reduced ligand-induced Ca+ 2 signaling, invasion of TNBC into Matrigel, and transwell migration. Leronlimab enhanced the BCa cell killing of the BCa chemotherapy reagent, doxorubicin. In xenografts conducted with Nu/Nu mice, leronlimab reduced lung metastasis of the TNBC cell line, MB-MDA-231, by > 98% at 6 weeks. Treatment with leronlimab reduced the metastatic tumor burden of established TNBC lung metastasis. CONCLUSIONS: The safety profile of leronlimab, together with strong preclinical evidence to both prevent and reduce established breast cancer metastasis herein, suggests studies of clinical efficacy may be warranted.

An Update on Glioblastoma Biology, Genetics, and Current Therapies: Novel Inhibitors of the G Protein-Coupled Receptor CCR5.

The mechanisms governing therapeutic resistance of the most aggressive and lethal primary brain tumor in adults, glioblastoma, have increasingly focused on tumor stem cells. These cells, protected by the periarteriolar hypoxic GSC niche, contribute to the poor efficacy of standard of care treatment of glioblastoma. Integrated proteogenomic and metabolomic analyses of glioblastoma tissues and single cells have revealed insights into the complex heterogeneity of glioblastoma and stromal cells, comprising its tumor microenvironment (TME). An additional factor, which isdriving poor therapy response is the distinct genetic drivers in each patient's tumor, providing the rationale for a more individualized or personalized approach to treatment. We recently reported that the G protein-coupled receptor CCR5, which contributes to stem cell expansion in other cancers, is overexpressed in glioblastoma cells. Overexpression of the CCR5 ligand CCL5 (RANTES) in glioblastoma completes a potential autocrine activation loop to promote tumor proliferation and invasion. CCL5 was not expressed in glioblastoma stem cells, suggesting a need for paracrine activation of CCR5 signaling by the stromal cells. TME-associated immune cells, such as resident microglia, infiltrating macrophages, T cells, and mesenchymal stem cells, possibly release CCR5 ligands, providing heterologous signaling between stromal and glioblastoma stem cells. Herein, we review current therapies for glioblastoma, the role of CCR5 in other cancers, and the potential role for CCR5 inhibitors in the treatment of glioblastoma.

A cyclin D1 intrinsically disordered domain accesses modified histone motifs to govern gene transcription.

The essential G1-cyclin, CCND1, is frequently overexpressed in cancer, contributing to tumorigenesis by driving cell-cycle progression. D-type cyclins are rate-limiting regulators of G1-S progression in mammalian cells via their ability to bind and activate CDK4 and CDK6. In addition, cyclin D1 conveys kinase-independent transcriptional functions of cyclin D1. Here we report that cyclin D1 associates with H2BS14 via an intrinsically disordered domain (IDD). The same region of cyclin D1 was necessary for the induction of aneuploidy, induction of the DNA damage response, cyclin D1-mediated recruitment into chromatin, and CIN gene transcription. In response to DNA damage H2BS14 phosphorylation occurs, resulting in co-localization with γH2AX in DNA damage foci. Cyclin D1 ChIP seq and γH2AX ChIP seq revealed ~14% overlap. As the cyclin D1 IDD functioned independently of the CDK activity to drive CIN, the IDD domain may provide a rationale new target to complement CDK-extinction strategies.

Multi-omic profiling of clear cell renal cell carcinoma identifies metabolic reprogramming associated with disease progression.

Clear cell renal cell carcinoma (ccRCC) is a complex disease with remarkable immune and metabolic heterogeneity. Here we perform genomic, transcriptomic, proteomic, metabolomic and spatial transcriptomic and metabolomic analyses on 100 patients with ccRCC from the Tongji Hospital RCC (TJ-RCC) cohort. Our analysis identifies four ccRCC subtypes including De-clear cell differentiated (DCCD)-ccRCC, a subtype with distinctive metabolic features. DCCD cancer cells are characterized by fewer lipid droplets, reduced metabolic activity, enhanced nutrient uptake capability and a high proliferation rate, leading to poor prognosis. Using single-cell and spatial trajectory analysis, we demonstrate that DCCD is a common mode of ccRCC progression. Even among stage I patients, DCCD is associated with worse outcomes and higher recurrence rate, suggesting that it cannot be cured by nephrectomy alone. Our study also suggests a treatment strategy based on subtype-specific immune cell infiltration that could guide the clinical management of ccRCC.

The Role and Therapeutic Targeting of CCR5 in Breast Cancer.

The G-protein-coupled receptor C-C chemokine receptor 5 (CCR5) functions as a co-receptor for the entry of HIV into immune cells. CCR5 binds promiscuously to a diverse array of ligands initiating cell signaling that includes guided migration. Although well known to be expressed on immune cells, recent studies have shown the induction of CCR5 on the surface of breast cancer epithelial cells. The function of CCR5 on breast cancer epithelial cells includes the induction of aberrant cell survival signaling and tropism towards chemo attractants. As CCR5 is not expressed on normal epithelium, the receptor provides a potential useful target for therapy. Inhibitors of CCR5 (CCR5i), either small molecules (maraviroc, vicriviroc) or humanized monoclonal antibodies (leronlimab) have shown anti-tumor and anti-metastatic properties in preclinical studies. In early clinical studies, reviewed herein, CCR5i have shown promising results and evidence for effects on both the tumor and the anti-tumor immune response. Current clinical studies have therefore included combination therapy approaches with checkpoint inhibitors.

The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities. The DACH1 gene encodes a winged helix/Forkhead DNA-binding protein that competes for binding to FOXM1 sites. Herein, DACH1 gene deletion within the 13q21.31-q21.33 region occurs in up to 18% of human PCa and was associated with increased AR activity and poor prognosis. In prostate OncoMice, prostate-specific deletion of the Dach1 gene enhanced prostatic intraepithelial neoplasia (PIN), and was associated with increased TGFb activity and DNA damage. Reduced Dach1 increased DNA damage in response to genotoxic stresses. DACH1 was recruited to sites of DNA damage, augmenting recruitment of Ku70/Ku80. Reduced Dach1 expression was associated with increased homology directed repair and resistance to PARP inhibitors and TGFb kinase inhibitors. Reduced Dach1 expression may define a subclass of PCa that warrants specific therapies.

The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities. The DACH1 gene encodes a winged helix/Forkhead DNA-binding protein that competes for binding to FOXM1 sites. Herein, DACH1 gene deletion within the 13q21.31-q21.33 region occurs in up to 18% of human PCa and was associated with increased AR activity and poor prognosis. In prostate OncoMice, prostate-specific deletion of the Dach1 gene enhanced prostatic intraepithelial neoplasia (PIN), and was associated with increased TGFß activity and DNA damage. Reduced Dach1 increased DNA damage in response to genotoxic stresses. DACH1 was recruited to sites of DNA damage, augmenting recruitment of Ku70/Ku80. Reduced Dach1 expression was associated with increased homology directed repair and resistance to PARP inhibitors and TGFß kinase inhibitors. Reduced Dach1 expression may define a subclass of PCa that warrants specific therapies.

Cell fate determination factor Dachshund reprograms breast cancer stem cell function.

The cell fate determination factor Dachshund was cloned as a dominant inhibitor of the hyperactive epidermal growth factor receptor ellipse. The expression of Dachshund is lost in human breast cancer associated with poor prognosis. Breast tumor-initiating cells (TIC) may contribute to tumor progression and therapy resistance. Here, endogenous DACH1 was reduced in breast cancer cell lines with high expression of TIC markers and in patient samples of the basal breast cancer phenotype. Re-expression of DACH1 reduced new tumor formation in serial transplantations in vivo, reduced mammosphere formation, and reduced the proportion of CD44(high)/CD24(low) breast tumor cells. Conversely, lentiviral shRNA to DACH1 increased the breast (B)TIC population. Genome-wide expression studies of mammary tumors demonstrated DACH1 repressed a molecular signature associated with stem cells (SOX2, Nanog, and KLF4) and genome-wide ChIP-seq analysis identified DACH1 binding to the promoter of the Nanog, KLF4, and Lin28 genes. KLF4/c-Myc and Oct4/Sox2 antagonized DACH1 repression of BTIC. Mechanistic studies demonstrated DACH1 directly repressed the Nanog and Sox2 promoters via a conserved domain. Endogenous DACH1 regulates BTIC in vitro and in vivo.

p21CIP1 attenuates Ras- and c-Myc-dependent breast tumor epithelial mesenchymal transition and cancer stem cell-like gene expression in vivo.

p21(CIP1/WAF1) is a downstream effector of tumor suppressors and functions as a cyclin-dependent kinase inhibitor to block cellular proliferation. Breast tumors may derive from self-renewing tumor-initiating cells (BT-ICs), which contribute to tumor progression, recurrence, and therapy resistance. The role of p21(CIP1) in regulating features of tumor stem cells in vivo is unknown. Herein, deletion of p21(CIP1), which enhanced the rate of tumorigenesis induced by mammary-targeted Ha-Ras or c-Myc, enhanced gene expression profiles and immunohistochemical features of epithelial mesenchymal transition (EMT) and putative cancer stem cells in vivo. Silencing of p21(CIP1) enhanced, and expression of p21(CIP1) repressed, features of EMT in transformed immortal human MEC lines. p21(CIP1) attenuated oncogene-induced BT-IC and mammosphere formation. Thus, the in vitro cell culture assays reflect the changes observed in vivo in transgenic mice. These findings establish a link between the loss of p21(CIP1) and the acquisition of breast cancer EMT and stem cell properties in vivo.

Assays for the Spectrum of Circulating Tumor Cells.

Cancer cells sharing stem cell properties are called "cancer stem cells" (CSCs). CSCs have distinct metabolic properties, are intrinsically drug resistant evading chemotherapies, are regulated by miRNA networks and participate in tumor relapse and metastases. During metastatic dissemination, circulating tumor cells (CTCs) invade distant organs and settle in supportive niches. In this process, the stem cell-like properties within CTCs contribute to CTC survival and eventually seed the growth of a secondary tumor. We herein describe methodologies for the analysis of CTCs as they reside in distinct functional pools with distinct characteristics.

The Renaissance of CDK Inhibitors in Breast Cancer Therapy: An Update on Clinical Trials and Therapy Resistance.

Cyclin-dependent kinases (CDKs) govern cell-cycle checkpoint transitions necessary for cancer cell proliferation. Recent developments have illustrated nuanced important differences between mono CDK inhibitor (CDKI) treatment and the combination therapies of breast cancers. The CDKIs that are currently FDA-approved for breast cancer therapy are oral agents that selectively inhibit CDK4 and CDK6, include palbociclib (Ibrance), ribociclib (Kisqali), and abemaciclib (Verzenio). CDKI therapy is effective in hormone receptor positive (HR+), and human epidermal growth factor receptor two negative (HER2-) advanced breast cancers (ABC) malignancies, but remains susceptible due to estrogen and progesterone receptor overexpression. Adding a CDK4/6I to endocrine therapy increases efficacy and delays disease progression. Given the side effects of CDKI, identifying potential new treatments to enhance CDKI effectiveness is essential. Recent long-term studies with Palbociclib, including the PALLAS and PENELOPE B, which failed to meet their primary endpoints of influencing progression-free survival, suggest a deeper mechanistic understanding of cyclin/CDK functions is required. The impact of CDKI on the anti-tumor immune response represents an area of great promise. CDKI therapy resistance that arises provides the opportunity for specific types of new therapies currently in clinical trials.

Acetylation of nuclear receptors in health and disease: an update.

Lysine acetylation is a common reversible post-translational modification of proteins that plays a key role in regulating gene expression. Nuclear receptors (NRs) include ligand-inducible transcription factors and orphan receptors for which the ligand is undetermined, which together regulate the expression of genes involved in development, metabolism, homeostasis, reproduction and human diseases including cancer. Since the original finding that the ERα, AR and HNF4 are acetylated, we now understand that the vast majority of NRs are acetylated and that this modification has profound effects on NR function. Acetylation sites are often conserved and involve both ordered and disordered regions of NRs. The acetylated residues function as part of an intramolecular signalling platform intersecting phosphorylation, methylation and other modifications. Acetylation of NR has been shown to impact recruitment into chromatin, co-repressor and coactivator complex formation, sensitivity and specificity of regulation by ligand and ligand antagonists, DNA binding, subcellular distribution and transcriptional activity. A growing body of evidence in mice indicates a vital role for NR acetylation in metabolism. Additionally, mutations of the NR acetylation site occur in human disease. This review focuses on the role of NR acetylation in coordinating signalling in normal physiology and disease.

c-Jun induces mammary epithelial cellular invasion and breast cancer stem cell expansion.

The molecular mechanisms governing breast tumor cellular self-renewal contribute to breast cancer progression and therapeutic resistance. The ErbB2 oncogene is overexpressed in approximately 30% of human breast cancers. c-Jun, the first cellular proto-oncogene, is overexpressed in human breast cancer. However, the role of endogenous c-Jun in mammary tumor progression is unknown. Herein, transgenic mice expressing the mammary gland-targeted ErbB2 oncogene were crossed with c-jun(f/f) transgenic mice to determine the role of endogenous c-Jun in mammary tumor invasion and stem cell function. The excision of c-jun by Cre recombinase reduced cellular migration, invasion, and mammosphere formation of ErbB2-induced mammary tumors. Proteomic analysis identified a subset of secreted proteins (stem cell factor (SCF) and CCL5) induced by ErbB2 expression that were dependent upon endogenous c-Jun expression. SCF and CCL5 were identified as transcriptionally induced by c-Jun. CCL5 rescued the c-Jun-deficient breast tumor cellular invasion phenotype. SCF rescued the c-Jun-deficient mammosphere production. Endogenous c-Jun thus contributes to ErbB2-induced mammary tumor cell invasion and self-renewal.

Evidence for DeltapH surface component (DeltapH(S)) of proton motive force in ATP synthesis of mitochondria.

BACKGROUND: One of the central debates in membrane bioenergetics is whether proton-dependent energy coupling mechanisms are mediated exclusively by protonic transmembrane electrochemical potentials, as delocalized pmf, DeltamicroH(+), or by more localized membrane surface proton pathways, as interfacial pmf, DeltamicroH(S). METHODS: We measure pH(S) in rat liver mitoplasts energized by respiration or ATP hydrolysis by inserting pH sensitive fluorescein-phosphatidyl-ethanolamine(F-PE) into mitoplast surface. RESULTS: In the presence of rotenone and Ap5A, succinate oxidation induces a bi-phasic interfacial protonation on the mitoplast membranes, a fast phase followed by a slow one, and an interfacial pH decrease of 0.5 to 0.9 pH units of mitoplast with no simultaneous pH changes in the bulk. Antimycin A, other inhibitors or uncouplers of mitochondrial respiration prevent the decrease of mitoplast pH(S), supporting that DeltamicroH(S) is dependent and controlled by energization of mitoplast membranes. A quantitative assay of ATP synthesis coupled with pH(S) of mitoplasts oxidizing succinate with malonate titration shows a parallel correlation between ATP synthesis, State 4 respiration and pH(S), but not with Psi(E). GENERAL SIGNIFICANCE: Our data substantiate pH(S) as the primary energy source of pmf for mitochondrial ATP synthesis. Evidence and discussion concerning the relative importance and interplay of pH(S) and Psi(E) in mitochondrial bioenergetics are also presented.

Dachshund inhibits oncogene-induced breast cancer cellular migration and invasion through suppression of interleukin-8.

Oncogene-mediated signaling to the host environment induces a subset of cytokines and chemokines. The Drosophila Dac gene promotes migration of the morphogenetic furrow during eye development. Expression of the cell-fate determination factor Dachshund (DACH1) was lost in poor prognosis invasive breast cancer. Mouse embryo fibroblasts derived from Dach1(-/-) mice demonstrated endogenous Dach1 constitutively represses cellular migration. DACH1 inhibited cellular migration and invasion of oncogene (Ras, Myc, ErbB2, c-Raf)-transformed human breast epithelial cells. An unbiased proteomic analysis identified and immunoneutralizing antibody and reconstitution experiments demonstrated IL-8 is a critical target of DACH1 mediating breast cancer cellular migration and metastasis in vivo. DACH1 bound the endogenous IL-8 promoter in ChIP assays and repressed the IL-8 promoter through the AP-1 and NF-kappaB binding sites. Collectively, our data identify a pathway by which an endogenous cell-fate determination factor blocks oncogene-dependent tumor metastasis via a key heterotypic mediator.

Mechanisms Governing Metabolic Heterogeneity in Breast Cancer and Other Tumors.

Reprogramming of metabolic priorities promotes tumor progression. Our understanding of the Warburg effect, based on studies of cultured cancer cells, has evolved to a more complex understanding of tumor metabolism within an ecosystem that provides and catabolizes diverse nutrients provided by the local tumor microenvironment. Recent studies have illustrated that heterogeneous metabolic changes occur at the level of tumor type, tumor subtype, within the tumor itself, and within the tumor microenvironment. Thus, altered metabolism occurs in cancer cells and in the tumor microenvironment (fibroblasts, immune cells and fat cells). Herein we describe how these growth advantages are obtained through either "convergent" genetic changes, in which common metabolic properties are induced as a final common pathway induced by diverse oncogene factors, or "divergent" genetic changes, in which distinct factors lead to subtype-selective phenotypes and thereby tumor heterogeneity. Metabolic heterogeneity allows subtyping of cancers and further metabolic heterogeneity occurs within the same tumor mass thought of as "microenvironmental metabolic nesting". Furthermore, recent findings show that mutations of metabolic genes arise in the majority of tumors providing an opportunity for the development of more robust metabolic models of an individual patient's tumor. The focus of this review is on the mechanisms governing this metabolic heterogeneity in breast cancer.

Pparγ1 Facilitates ErbB2-Mammary Adenocarcinoma in Mice.

HER2, which is associated with clinically aggressive disease, is overexpressed in 15-20% of breast cancers (BC). The host immune system participates in the therapeutic response of HER2+ breast cancer. Identifying genetic programs that participate in ErbB2-induced tumors may provide the rational basis for co-extinction therapeutic approaches. Peroxisome proliferator-activated receptor γ (PPARγ), which is expressed in a variety of malignancies, governs biological functions through transcriptional programs. Herein, genetic deletion of endogenous Pparγ1 restrained mammary tumor progression, lipogenesis, and induced local mammary tumor macrophage infiltration, without affecting other tissue hematopoietic stem cell pools. Endogenous Pparγ1 induced expression of both an EphA2-Amphiregulin and an inflammatory INFγ and Cxcl5 signaling module, that was recapitulated in human breast cancer. Pparγ1 bound directly to growth promoting and proinflammatory target genes in the context of chromatin. We conclude Pparγ1 promotes ErbB2-induced tumor growth and inflammation and represents a relevant target for therapeutic coextinction. Herein, endogenous Pparγ1 promoted ErbB2-mediated mammary tumor onset and progression. PPARγ1 increased expression of an EGF-EphA2 receptor tyrosine kinase module and a cytokine/chemokine 1 transcriptional module. The induction of a pro-tumorigenic inflammatory state by Pparγ1 may provide the rationale for complementary coextinction programs in ErbB2 tumors.

Nuclear factor-kappaB enhances ErbB2-induced mammary tumorigenesis and neoangiogenesis in vivo.

The (HER2/Neu) ErbB2 oncogene is commonly overexpressed in human breast cancer and is sufficient for mammary tumorigenesis in transgenic mice. Nuclear factor (NF)-kappaB activity is increased in both human and murine breast tumors. The immune response to mammary tumorigenesis may regulate tumor progression. The role of endogenous mammary epithelial cell NF-kappaB had not previously been determined in immune-competent animals. Furthermore, the role of the NF-kappaB components, p50 and p65, in tumor growth was not known. Herein, the expression of a stabilized form of the NF-kappaB-inhibiting IkappaBalpha protein (IkappaBalphaSR) in breast tumor cell lines that express oncogenic ErbB2 inhibited DNA synthesis and growth in both two- and three-dimensional cultures. Either NF-kappaB inhibition or selective silencing of p50 or p65 led to a loss of contact-independent tumor growth in vitro. IkappaBalphaSR reversed the features of the oncogene-induced phenotype under three-dimensional growth conditions. The NF-kappaB blockade inhibited ErbB2-induced mammary tumor growth in both immune-competent and immune-deficient mice. These findings were associated with both reduced tumor microvascular density and a reduction in the amount of vascular endothelial growth factor. The expression of IkappaBalphaSR in breast cancer tumors inhibited angiogenesis. Thus, mammary epithelial cell NF-kappaB activity enhances ErbB2-mediated mammary tumorigenesis in vivo by promoting both growth and survival signaling via the promotion of tumor vasculogenesis.

Somatic excision demonstrates that c-Jun induces cellular migration and invasion through induction of stem cell factor.

Cancer cells arise through sequential acquisition of mutations in tumor suppressors and oncogenes. c-Jun, a critical component of the AP-1 complex, is frequently overexpressed in diverse tumor types and has been implicated in promoting cellular proliferation, migration, and angiogenesis. Functional analysis of candidate genetic targets using germ line deletion in murine models can be compromised through compensatory mechanisms. As germ line deletion of c-jun induces embryonic lethality, somatic deletion of the c-jun gene was conducted using floxed c-jun (c-jun(f/f)) conditional knockout mice. c-jun-deleted cells showed increased cellular adhesion, stress fiber formation, and reduced cellular migration. The reduced migratory velocity and migratory directionality was rescued by either c-Jun reintroduction or addition of secreted factors from wild-type cells. An unbiased analysis of cytokines and growth factors, differentially expressed and showing loss of secretion upon c-jun deletion, identified stem cell factor (SCF) as a c-Jun target gene. Immunoneutralizing antibody to SCF reduced migration of wild-type cells. SCF addition rescued the defect in cellular adhesion, cellular velocity, directional migration, transwell migration, and cellular invasion of c-jun(-/-) cells. c-Jun induced SCF protein, mRNA, and promoter activity. Induction of the SCF promoter required the c-Jun DNA-binding domain. c-Jun bound to the SCF promoter in chromatin immunoprecipitation assays. Mutation of the c-Jun binding site abolished c-Jun-mediated induction of the SCF promoter. These studies demonstrate an essential role of c-Jun in cellular migration through induction of SCF.

Cell fate determination factor DACH1 inhibits c-Jun-induced contact-independent growth.

The cell fate determination factor DACH1 plays a key role in cellular differentiation in metazoans. DACH1 is engaged in multiple context-dependent complexes that activate or repress transcription. DACH1 can be recruited to DNA via the Six1/Eya bipartite transcription (DNA binding/coactivator) complex. c-Jun is a critical component of the activator protein (AP)-1 transcription factor complex and can promote contact-independent growth. Herein, DACH1 inhibited c-Jun-induced DNA synthesis and cellular proliferation. Excision of c-Jun with Cre recombinase, in c-jun(f1/f1) 3T3 cells, abrogated DACH1-mediated inhibition of DNA synthesis. c-Jun expression rescued DACH1-mediated inhibition of cellular proliferation. DACH1 inhibited induction of c-Jun by physiological stimuli and repressed c-jun target genes (cyclin A, beta-PAK, and stathmin). DACH1 bound c-Jun and inhibited AP-1 transcriptional activity. c-jun and c-fos were transcriptionally repressed by DACH1, requiring the conserved N-terminal (dac and ski/sno [DS]) domain. c-fos transcriptional repression by DACH1 requires the SRF site of the c-fos promoter. DACH1 inhibited c-Jun transactivation through the delta domain of c-Jun. DACH1 coprecipitated the histone deacetylase proteins (HDAC1, HDAC2, and NCoR), providing a mechanism by which DACH1 represses c-Jun activity through the conserved delta domain. An oncogenic v-Jun deleted of the delta domain was resistant to DACH1 repression. Collectively, these studies demonstrate a novel mechanism by which DACH1 blocks c-Jun-mediated contact-independent growth through repressing the c-Jun delta domain.