GATA2 facilitates steroid receptor coactivator recruitment to the androgen receptor complex.

The androgen receptor (AR) is a key driver of prostate cancer (PC), even in the state of castration-resistant PC (CRPC) and frequently even after treatment with second-line hormonal therapies such as abiraterone and enzalutamide. The persistence of AR activity via both ligand-dependent and ligand-independent mechanisms (including constitutively active AR splice variants) highlights the unmet need for alternative approaches to block AR signaling in CRPC. We investigated the transcription factor GATA-binding protein 2 (GATA2) as a regulator of AR signaling and an actionable therapeutic target in PC. We demonstrate that GATA2 directly promotes expression of both full-length and splice-variant AR, resulting in a strong positive correlation between GATA2 and AR expression in both PC cell lines and patient specimens. Conversely, GATA2 expression is repressed by androgen and AR, suggesting a negative feedback regulatory loop that, upon androgen deprivation, derepresses GATA2 to contribute to AR overexpression in CRPC. Simultaneously, GATA2 is necessary for optimal transcriptional activity of both full-length and splice-variant AR. GATA2 colocalizes with AR and Forkhead box protein A1 on chromatin to enhance recruitment of steroid receptor coactivators and formation of the transcriptional holocomplex. In agreement with these important functions, high GATA2 expression and transcriptional activity predicted worse clinical outcome in PC patients. A GATA2 small molecule inhibitor suppressed the expression and transcriptional function of both full-length and splice-variant AR and exerted potent anticancer activity against PC cell lines. We propose pharmacological inhibition of GATA2 as a first-in-field approach to target AR expression and function and improve outcomes in CRPC.

Prostate cancer-associated mutations in speckle-type POZ protein (SPOP) regulate steroid receptor coactivator 3 protein turnover.

The p160 steroid receptor coactivators (SRCs) SRC-1, SRC-2 [nuclear receptor coactivator (NCOA)2], and SRC-3 [amplified in breast cancer 1 (AIB1)/NCOA3] are key pleiotropic "master regulators" of transcription factor activity necessary for cancer cell proliferation, survival, metabolism, and metastasis. SRC overexpression and overactivation occur in numerous human cancers and are associated with poor clinical outcomes and resistance to therapy. In prostate cancer (PC), the p160 SRCs play critical roles in androgen receptor transcriptional activity, cell proliferation, and resistance to androgen deprivation therapy. We recently demonstrated that the E3 ubiquitin ligase adaptor speckle-type poxvirus and zinc finger (POZ) domain protein (SPOP) interacts directly with SRC-3 and promotes its cullin 3-dependent ubiquitination and proteolysis in breast cancer, thus functioning as a potential tumor suppressor. Interestingly, somatic heterozygous missense mutations in the SPOP substrate-binding cleft recently were identified in up to 15% of human PCs (making SPOP the gene most commonly affected by nonsynonymous point mutations in PC), but their contribution to PC pathophysiology remains unknown. We now report that PC-associated SPOP mutants cannot interact with SRC-3 protein or promote its ubiquitination and degradation. Our data suggest that wild-type SPOP plays a critical tumor suppressor role in PC cells, promoting the turnover of SRC-3 protein and suppressing androgen receptor transcriptional activity. This tumor suppressor effect is abrogated by the PC-associated SPOP mutations. These studies provide a possible explanation for the role of SPOP mutations in PC, and highlight the potential of SRC-3 as a therapeutic target in PC.

Epigenetic alteration by DNA-demethylating treatment restores apoptotic response to glucocorticoids in dexamethasone-resistant human malignant lymphoid cells.

BACKGROUND: Glucocorticoids (GCs) are often included in the therapy of lymphoid malignancies because they kill several types of malignant lymphoid cells. GCs activate the glucocorticoid receptor (GR), to regulate a complex genetic network, culminating in apoptosis. Normal lymphoblasts and many lymphoid malignancies are sensitive to GC-driven apoptosis. Resistance to GCs can be a significant clinical problem, however, and correlates with resistance to several other major chemotherapeutic agents. METHODS: We analyzed the effect of treatment with the cytosine analogue 5 aza-2' deoxycytidine (AZA) on GC resistance in two acute lymphoblastic leukemia (T or pre-T ALL) cell lines- CEM and Molt-4- and a (B-cell) myeloma cell line, RPMI 8226. Methods employed included tissue culture, flow cytometry, and assays for clonogenicity, cytosine extension, immunochemical identification of proteins, and gene transactivation. High throughput DNA sequencing was used to confirm DNA methylation status. CONCLUSIONS: Treatment of these cells with AZA resulted in altered DNA methylation and restored GC-evoked apoptosis in all 3 cell lines. In CEM cells the altered epigenetic state resulted in site-specific phosphorylation of the GR, increased GR potency, and GC-driven induction of the GR from promoters that lie in CpG islands. In RPMI 8226 cells, expression of relevant coregulators of GR function was altered. Activation of p38 mitogen-activated protein kinase (MAPK), which is central to a feed-forward mechanism of site-specific GR phosphorylation and ultimately, apoptosis, occurred in all 3 cell lines. These data show that in certain malignant hematologic B- and T-cell types, epigenetically controlled GC resistance can be reversed by cell exposure to a compound that causes DNA demethylation. The results encourage studies of application to in vivo systems, looking towards eventual clinical applications.

SPOP Mutations Target STING1 Signaling in Prostate Cancer and Create Therapeutic Vulnerabilities to PARP Inhibitor-Induced Growth Suppression.

PURPOSE: Speckle-type POZ protein (SPOP) is important in DNA damage response (DDR) and maintenance of genomic stability. Somatic heterozygous missense mutations in the SPOP substrate-binding cleft are found in up to 15% of prostate cancers. While mutations in SPOP predict for benefit from androgen receptor signaling inhibition (ARSi) therapy, outcomes for patients with SPOP-mutant (SPOPmut) prostate cancer are heterogeneous and targeted treatments for SPOPmut castrate-resistant prostate cancer (CRPC) are lacking. EXPERIMENTAL DESIGN: Using in silico genomic and transcriptomic tumor data, proteomics analysis, and genetically modified cell line models, we demonstrate mechanistic links between SPOP mutations, STING signaling alterations, and PARP inhibitor vulnerabilities. RESULTS: We demonstrate that SPOP mutations are associated with upregulation of a 29-gene noncanonical (NC) STING (NC-STING) signature in a subset of SPOPmut, treatment-refractory CRPC patients. We show in preclinical CRPC models that SPOP targets and destabilizes STING1 protein, and prostate cancer-associated SPOP mutations result in upregulated NC-STING-NF-κB signaling and macrophage- and tumor microenvironment (TME)-facilitated reprogramming, leading to tumor cell growth. Importantly, we provide in vitro and in vivo mechanism-based evidence that PARP inhibitor (PARPi) treatment results in a shift from immunosuppressive NC-STING-NF-κB signaling to antitumor, canonical cGAS-STING-IFNß signaling in SPOPmut CRPC and results in enhanced tumor growth inhibition. CONCLUSIONS: We provide evidence that SPOP is critical in regulating immunosuppressive versus antitumor activity downstream of DNA damage-induced STING1 activation in prostate cancer. PARPi treatment of SPOPmut CRPC alters this NC-STING signaling toward canonical, antitumor cGAS-STING-IFNß signaling, highlighting a novel biomarker-informed treatment strategy for prostate cancer.

ATR Inhibition Induces CDK1-SPOP Signaling and Enhances Anti-PD-L1 Cytotoxicity in Prostate Cancer.

PURPOSE: Despite significant benefit for other cancer subtypes, immune checkpoint blockade (ICB) therapy has not yet been shown to significantly improve outcomes for men with castration-resistant prostate cancer (CRPC). Prior data have shown that DNA damage response (DDR) deficiency, via genetic alteration and/or pharmacologic induction using DDR inhibitors (DDRi), may improve ICB response in solid tumors in part due to induction of mitotic catastrophe and innate immune activation. Discerning the underlying mechanisms of this DDRi-ICB interaction in a prostate cancer-specific manner is vital to guide novel clinical trials and provide durable clinical responses for men with CRPC. EXPERIMENTAL DESIGN: We treated prostate cancer cell lines with potent, specific inhibitors of ATR kinase, as well as with PARP inhibitor, olaparib. We performed analyses of cGAS-STING and DDR signaling in treated cells, and treated a syngeneic androgen-indifferent, prostate cancer model with combined ATR inhibition and anti-programmed death ligand 1 (anti-PD-L1), and performed single-cell RNA sequencing analysis in treated tumors. RESULTS: ATR inhibitor (ATRi; BAY1895433) directly repressed ATR-CHK1 signaling, activated CDK1-SPOP axis, leading to destabilization of PD-L1 protein. These effects of ATRi are distinct from those of olaparib, and resulted in a cGAS-STING-initiated, IFN-ß-mediated, autocrine, apoptotic response in CRPC. The combination of ATRi with anti-PD-L1 therapy resulted in robust innate immune activation and a synergistic, T-cell-dependent therapeutic response in our syngeneic mouse model. CONCLUSIONS: This work provides a molecular mechanistic rationale for combining ATR-targeted agents with immune checkpoint blockade for patients with CRPC. Multiple early-phase clinical trials of this combination are underway.

PARP and CDK4/6 Inhibitor Combination Therapy Induces Apoptosis and Suppresses Neuroendocrine Differentiation in Prostate Cancer.

We analyzed the efficacy and mechanistic interactions of PARP inhibition (PARPi; olaparib) and CDK4/6 inhibition (CDK4/6i; palbociclib or abemaciclib) combination therapy in castration-resistant prostate cancer (CRPC) and neuroendocrine prostate cancer (NEPC) models. We demonstrated that combined olaparib and palbociblib or abemaciclib treatment resulted in synergistic suppression of the p-Rb1-E2F1 signaling axis at the transcriptional and posttranslational levels, leading to disruption of cell-cycle progression and inhibition of E2F1 gene targets, including genes involved in DDR signaling/damage repair, antiapoptotic BCL-2 family members (BCL-2 and MCL-1), CDK1, and neuroendocrine differentiation (NED) markers in vitro and in vivo In addition, olaparib + palbociclib or olaparib + abemaciclib combination treatment resulted in significantly greater growth inhibition and apoptosis than either single agent alone. We further showed that PARPi and CDK4/6i combination treatment-induced CDK1 inhibition suppressed p-S70-BCL-2 and increased caspase cleavage, while CDK1 overexpression effectively prevented the downregulation of p-S70-BCL-2 and largely rescued the combination treatment-induced cytotoxicity. Our study defines a novel combination treatment strategy for CRPC and NEPC and demonstrates that combination PARPi and CDK4/6i synergistically promotes suppression of the p-Rb1-E2F1 axis and E2F1 target genes, including CDK1 and NED proteins, leading to growth inhibition and increased apoptosis in vitro and in vivo Taken together, our results provide a molecular rationale for PARPi and CDK4/6i combination therapy and reveal mechanism-based clinical trial opportunities for men with NEPC.

Use of recombinant cell-permeable small peptides to modulate glucocorticoid sensitivity of acute lymphoblastic leukemia cells.

Glucocorticoid (GC) hormones induce apoptosis in T-cell and pre-B-cell acute lymphoblastic leukemia (ALL) cells. Steroid-mediated apoptosis requires a threshold level of the glucocorticoid receptor (GR) protein, and increasing the intracellular GR levels in ALL cells would augment their hormone sensitivity. A protein transduction domain (PTD) approach was used to accomplish this. We produced an HIV Tat PTD domain fusion protein (Tat-GR(554-777)) that potentially competes for the degradation of GR protein by the ubiquitin-proteasome system and should thus increase its intracellular levels by "stabilizing" the GR. We also designed a fusion peptide for the c-Myb DNA binding domain, Tat-c-Myb DBD, since the biological function of this peptide as a dominant negative inhibitor of the c-Myb protein was already known. Purified, bacterially expressed Tat-c-Myb DBD and Tat-GR(554-777) exhibited highly efficient transduction into cultured ALL cell lines including 697 (pre-B-ALL) and CEM-C7 (T-ALL) cells. As expected, the transduced Tat-c-Myb DBD peptide inhibited steroid-mediated stimulation of a GR promoter-luciferase reporter gene. Significantly, transduced Tat-GR(554-777) effectively increased intracellular GR levels in the GC-resistant T-ALL cell line, CEM-C1, and in the pre-B-ALL 697 cell line. Furthermore, transduction of Tat-GR(554-777) rendered GC-resistant CEM-C1 cells sensitive to steroid killing and further sensitized 697 cells to steroid. The use of Tat-fusion peptide transduction may eventually lead to innovative therapeutic modalities to improve the clinical response of patients suffering from T-cell and pre-B-cell acute lymphoblastic leukemia by increasing steroid responsiveness and perhaps converting steroid-resistant leukemia to a hormone-responsive phenotype.

A conserved molecular mechanism is responsible for the auto-up-regulation of glucocorticoid receptor gene promoters.

Glucocorticoid (GC) hormones are widely used in the treatment of acute lymphoblastic leukemia (ALL). Whereas a high level of GC receptor (GR) protein is associated with the sensitivity of ALL cells to steroid-mediated apoptosis, the auto-up-regulation of human (h)GR mRNA and protein is also found in hormone-sensitive ALL cell lines. We have characterized the hGR gene-proximal promoters for DNA sequences and transcription factors required for hormone responsiveness in T lymphoblasts. Sequences at -4559/-4525 and -2956/-2916, relative to the translation start site, function as strong composite GC response units (GRUs). Both GRUs include adjacent protein recognition sequences for the c-Myb transcription factor and the GR as a DNA cassette. An Ets-binding sequence overlaps the GR-binding site in the -4559/-4525 GRU, whereas an Ets-binding site present in the -2956/-2916 GRU does not overlap the GR/c-Myb-binding cassette. The Ets protein family member, PU.1, blocks hormonal activation of the -4559/-4525 GR/c-Myb-binding cassette but does not interfere with the responsiveness of the -2956/-2916 GRU. Thus, the hGR 1A GRU (described previously), the -4559/-4525 GRU, and the -2956/-2916 GRU have a similar structure and can mediate cell type-specific hormonal auto-up-regulation of hGR promoter activity in steroid-sensitive ALL cells. However, subtle differences in the GRU architecture result in differential sensitivity of the promoters to Ets family members such as PU.1. The architecture of the GRU and the spectrum of specific transcription factors present in different types of ALL might allow the development of a tailored therapy to enhance steroid sensitivity in ALL patients.

PARP Inhibition Suppresses GR-MYCN-CDK5-RB1-E2F1 Signaling and Neuroendocrine Differentiation in Castration-Resistant Prostate Cancer.

PURPOSE: In this study, we addressed the underlying mechanisms for the association between enzalutamide (ENZ) treatment and neuroendocrine prostate cancer (NEPC), and the critical involvement of MYCN, and loss of RB1 function in neuroendocrine differentiation (NED) of prostatic epithelial cells, and the development of NEPC. We further sought to determine whether PARP inhibition could suppress NEPC, and to identify molecular determinants of this therapeutic activity. EXPERIMENTAL DESIGN: We used a novel prostate cancer patient-derived xenograft (PDX) treatment model, prostatic adenocarcinoma and NEPC cell lines, an NEPC organoid line, and NEPC xenograft models to address the mechanistic basis of ENZ-induced NED, and to analyze suppression of NED and NEPC growth by PARP inhibition. RESULTS: We identified an ENZ treatment-associated glucocorticoid receptor (GR)-MYCN-CDK5-RB1-E2F1 signaling pathway that drives NED in prostatic adenocarcinoma PDX and cell line models. Mechanistically, long-term ENZ treatment transcriptionally upregulates signaling of the GR-MYCN axis, leading to CDK5R1 and CDK5R2 upregulation, Rb1 phosphorylation, and N-Myc-mediated and E2F1-mediated NED gene expression. Importantly, olaparib (OLA) or talazoparib (TALA) suppressed these activities, and the combination of OLA and dinaciclib (DINA), an inhibitor of CDK2 and CDK5, which also inhibits Rb1 phosphorylation, suppressed NED and significantly improved therapeutic efficiency in NEPC cells in vitro and in NEPC tumors in vivo. CONCLUSIONS: The results of our study indicate an important role of GR-MYCN-CDK5R1/2-RB1-NED signaling in ENZ-induced and PARP inhibitor-suppressed NEPC. We also demonstrated efficacy for OLA+DINA combination therapy in NEPC xenograft models.

Interaction between the interferon signaling pathway and the human glucocorticoid receptor gene 1A promoter.

The newly described 1A promoter of the human glucocorticoid receptor (hGR) gene contains an interferon (IFN) regulatory factor element (IRF-E), a binding motif for the family of proteins termed IFN regulatory factors (IRFs) that are regulated by IFNs. To examine the in vivo role of IFNs in hGR gene regulation, human T cell lines (CEM-C7 and Jurkat) were treated with IFN gamma. IFN gamma rapidly induces the expression of IRF-1 proteins in a dose- and time-dependent manner. Luciferase expression is induced by IFN treatment in Jurkat cells transfected with an hGR 1A promoter IRF-E/luciferase reporter gene, but induction is lost with deletion of the IRF-E. Electrophoretic mobility shift and supershift analyses indicate an increase in the binding of IRF-1 to oligonucleotides containing the hGR 1A promoter IRF-E after IFN gamma treatment, whereas IRF-2 binding to this oligonucleotide is unchanged. Human IRF-1 and IRF-2 proteins expressed in Chinese hamster ovary cells bind to the hGR 1A promoter IRF-E; however, only IRF-1 activates transcription. Although IFNs clearly activate a transfected reporter gene containing the hGR 1A promoter in T cells, they do not alter the sensitivity of CEM-C7 cells to glucocorticoid-induced apoptosis. Additional studies revealed that the glucocorticoid steroid hormone, dexamethasone (DEX), completely blocks IFN induction of IRF-1 mRNA levels. This could explain the lack of any greater apoptotic response to a combination of DEX plus IFN compared with the response to DEX alone. In addition, treatment with IFN gamma alone does not alter endogenous GR mRNA levels (including exon 1A-containing transcripts derived from the hGR 1A promoter) in T lymphoblast cells, even though IRF-1 levels are induced. The difference in IRF-1-driven transcription between the hGR 1A reporter construct and the endogenous hGR 1A promoter could potentially be due to epigenetic effects, such as methylation.

Molecular cloning and characterization of the catalytic domain of zebrafish homologue of the ataxia-telangiectasia mutated gene.

Inherited biallelic mutations of the ATM (ataxia-telangiectasia mutated) gene in humans cause ataxia-telangiectasia, a rare autosomal recessive disorder associated with progressive neuro-degeneration, cancer predisposition, immunodeficiency, and hypersensitivity to ionizing radiation. The ATM gene is highly conserved across a wide range of species. In an attempt to establish a zebrafish (Danio rerio) model of ataxia-telangiectasia, we cloned the coding sequence of the catalytic domain of the zebrafish homologue of ATM and found it to contain an open reading frame encoding 907 amino acids at the carboxyl terminus of the zebrafish ATM (zATM). The catalytic domain of zATM shares 67% and 66% homology with human ATM (hATM) and mouse ATM (mATM), respectively. The full-length mRNA encoding zATM is found to be approximately 11 kb by Northern hybridization, and the expression of zATM is observed in different adult and embryonic tissues. Overexpression of a kinase-inactive zATM domain in human cells has a dominant-negative effect against hATM function. Expression of the altered zATM in ZF4 cells leads to an A-T-like phenotype in response to ionizing radiation. These results taken together indicate that zATM is the homologue of hATM. Furthermore, using the kinase-inactive form of zATM should allow manipulation of zATM function in fish cells.

Steroid-responsive sequences in the human glucocorticoid receptor gene 1A promoter.

At least three promoters (1A, 1B, and 1C) control the expression of mRNA transcripts for the human glucocorticoid receptor (hGR) protein. An hGR 1A promoter/exon sequence (-218/+269) contains at least 12 deoxyribonuclease (DNase) I footprints that contain bound protein. Whereas four of these footprints (FP6, FP7, FP8, and FP11) contain bound hGR in protein-DNA complexes that are formed, only two (FP7 and FP11) appear to be important for the up-regulation of hGR 1A promoter/exon activity in T-lymphoblasts. Furthermore, the activity of these DNA elements depends upon the promoter context, leading to a redundant and complex regulation of expression of the hGR 1A promoter/exon. FP7 appears to be required for hormonal responsiveness in the absence of upstream sequences (+41/+191), whereas the hormonal responsiveness of FP11 requires a functional, adjacent FP12 DNA sequence. FP12 contains overlapping binding sites for the protooncogene transcription factors c-Myb and c-Ets. It seems likely that binding of either c-Myb or c-Ets to FP12 is necessary for the direct or indirect binding of the hGR to FP11 (a nonconsensus glucocorticoid response element), and the resultant steroid-responsiveness of the hGR 1A promoter/exon sequence. We propose that the identity of the accessory transcription factor bound to FP12 (c-Myb or c-Ets) may determine the nature of regulation (positive or negative) of hGR gene expression by hormone, and that this may be important for hormone-induced apoptosis in T cell acute lymphoblastic leukemia.

miR-137 Targets p160 Steroid Receptor Coactivators SRC1, SRC2, and SRC3 and Inhibits Cell Proliferation.

The p160 family of steroid receptor coactivators (SRCs) are pleiotropic transcription factor coactivators and "master regulators" of gene expression that promote cancer cell proliferation, survival, metabolism, migration, invasion, and metastasis. Cancers with high p160 SRC expression exhibit poor clinical outcomes and resistance to therapy, highlighting the SRCs as critical oncogenic drivers and, thus, therapeutic targets. microRNAs are important epigenetic regulators of protein expression. To examine the regulation of p160 SRCs by microRNAs, we used and combined 4 prediction algorithms to identify microRNAs that could target SRC1, SRC2, and SRC3 expression. For validation of these predictions, we assessed p160 SRC protein expression and cell viability after transfection of corresponding microRNA mimetics in breast cancer, uveal melanoma, and prostate cancer (PC) cell lines. Transfection of selected microRNA mimetics into breast cancer, uveal melanoma, and PC cells depleted SRC protein expression levels and exerted potent antiproliferative activity in these cell types. In particular, microRNA-137 (miR-137) depleted expression of SRC1, SRC2, and very potently, SRC3. The latter effect can be attributed to the presence of 3 miR-137 recognition sequences within the SRC3 3'-untranslated region. Using reverse phase protein array analysis, we identified a network of proteins, in addition to SRC3, that were modulated by miR-137 in PC cells. We also found that miR-137 and its host gene are epigenetically silenced in human cancer specimens and cell lines. These results support the development and testing of microRNA-based therapies (in particular based on restoring miR-137 levels) for targeting the oncogenic family of p160 SRCs in cancer.

Androgen receptor is the key transcriptional mediator of the tumor suppressor SPOP in prostate cancer.

Somatic missense mutations in the substrate-binding pocket of the E3 ubiquitin ligase adaptor SPOP are present in up to 15% of human prostate adenocarcinomas, but are rare in other malignancies, suggesting a prostate-specific mechanism of action. SPOP promotes ubiquitination and degradation of several protein substrates, including the androgen receptor (AR) coactivator SRC-3. However, the relative contributions that SPOP substrates may make to the pathophysiology of SPOP-mutant (mt) prostate adenocarcinomas are unknown. Using an unbiased bioinformatics approach, we determined that the gene expression profile of prostate adenocarcinoma cells engineered to express mt-SPOP overlaps greatly with the gene signature of both SRC-3 and AR transcriptional output, with a stronger similarity to AR than SRC-3. This finding suggests that in addition to its SRC-3-mediated effects, SPOP also exerts SRC-3-independent effects that are AR-mediated. Indeed, we found that wild-type (wt) but not prostate adenocarcinoma-associated mutants of SPOP promoted AR ubiquitination and degradation, acting directly through a SPOP-binding motif in the hinge region of AR. In support of these results, tumor xenografts composed of prostate adenocarcinoma cells expressing mt-SPOP exhibited higher AR protein levels and grew faster than tumors composed of prostate adenocarcinoma cells expressing wt-SPOP. Furthermore, genetic ablation of SPOP was sufficient to increase AR protein levels in mouse prostate. Examination of public human prostate adenocarcinoma datasets confirmed a strong link between transcriptomic profiles of mt-SPOP and AR. Overall, our studies highlight the AR axis as the key transcriptional output of SPOP in prostate adenocarcinoma and provide an explanation for the prostate-specific tumor suppressor role of wt-SPOP.

c-Myb interacts with the glucocorticoid receptor and regulates its level in pre-B-acute lymphoblastic leukemia cells.

Glucocorticoid (GC) hormones are used in the treatment of hematopoietic malignancies. When the GC binds to the glucocorticoid receptor (GR) protein, c-Myb and GR are recruited at the Glucocorticoid Response Unit in the DNA. Here we demonstrate that c-Myb interacts with the GR and that decreasing c-Myb amounts reduces the levels of GR transcripts and protein in 697 pre-B-acute lymphoblastic leukemia (ALL) cells. Furthermore, the auto-upregulation of GR promoter 1C and promoter 1D is blunted at reduced c-Myb levels. Taken together, these data show that c-Myb is a direct, key regulator of the GR. Unexpectedly, the reduction in c-Myb levels increased the sensitivity of the cells to steroid-mediated apoptosis. This was because the reduction in c-Myb itself decreases cell viability, and the residual GR remained above the threshold needed to trigger apoptosis. These studies show the mutual importance of c-Myb and the GR in controlling survival of pre-B ALL cells.

A new, lineage specific, autoup-regulation mechanism for human glucocorticoid receptor gene expression in 697 pre-B-acute lymphoblastic leukemia cells.

Glucocorticoid (GC) steroid hormones induce apoptosis in acute lymphoblastic leukemia (ALL). Autoup-regulation of human GC receptor (hGR) levels is associated with sensitivity to GC-mediated apoptosis. Among the major hGR promoters expressed in 697 pre-B-ALL cells (1A, 1B, 1C, and 1D), only promoters 1C and 1D are selectively activated by the hormone. Promoter 1B is unresponsive, and promoter 1A is down-regulated by dexamethasone (Dex) in 697 cells, whereas they are both up-regulated in CEM-C7 T-ALL cells. Autoup-regulation of promoter 1C and 1D in 697 cells requires sequences containing GC response units (GRUs) (1C GRU, -2915/-2956; 1D GRU, -4525/-4559) that were identified previously in CEM-C7 cells. These GRUs potentially bind GR, c-myeloblastosis (c-Myb), and E-twenty six (Ets) proteins; 697 cells express high levels of c-Myb protein, as well as the E-twenty six family protein members, PU.1 and Spi-B. Dex treatment in 697 cells elevates the expression of c-Myb and decreases levels of both Spi-B and PU.1. Chromatin immunoprecipitation assays revealed the specific recruitment of GR, c-Myb, and cAMP response element-binding protein binding protein to the 1C and 1D GRUs upon Dex treatment, correlating to observed autoup-regulated activity in these two promoters. These data suggest a hormone activated, lineage-specific mechanism to control the autoup-regulation of hGR gene expression in 697 pre-B-ALL cells via steroid-mediated changes in GR coregulator expression. These findings may be helpful in understanding the mechanism that determines the sensitivity of B-ALL leukemia cells to hormone-induced apoptosis.

c-Myb and members of the c-Ets family of transcription factors act as molecular switches to mediate opposite steroid regulation of the human glucocorticoid receptor 1A promoter.

Steroid auto-regulation of the human glucocorticoid receptor (hGR) 1A promoter in lymphoblast cells resides largely in two DNA elements (footprints 11 and 12). We show here that c-Myb and c-Ets family members (Ets-1/2, PU.1, and Spi-B) control hGR 1A promoter regulation in T- and B-lymphoblast cells. Two T-lymphoblast lines, CEM-C7 and Jurkat, contain high levels of c-Myb and low levels of PU.1, whereas the opposite is true in IM-9 B-lymphoblasts. In Jurkat cells, overexpression of c-Ets-1, c-Ets-2, or PU.1 effectively represses dexamethasone-mediated up-regulation of an hGR 1A promoter-luciferase reporter gene, as do dominant negative c-Myb (c-Myb DNA-binding domain) or Ets proteins (Ets-2 DNA-binding domain). Overexpression of c-Myb in IM-9 cells confers hormone-dependent up-regulation to the hGR 1A promoter reporter gene. Chromatin immunoprecipitation assays show that hormone treatment causes the recruitment of hGR and c-Myb to the hGR 1A promoter in CEM-C7 cells, whereas hGR and PU.1 are recruited to this promoter in IM-9 cells. These observations suggest that the specific transcription factor that binds to footprint 12, when hGR binds to the adjacent footprint 11, determines the direction of hGR 1A promoter auto-regulation. This leads to a "molecular switch" model for auto-regulation of the hGR 1A promoter.

Human glucocorticoid receptor alpha transcript splice variants with exon 2 deletions: evidence for tissue- and cell type-specific functions.

Alternative splicing of exon 9 in human glucocorticoid receptor (hGR) transcripts yields two native hGR transcripts and proteins, hGRalpha and hGRbeta. We have now identified four novel hGRalpha transcripts that have various deletions of exon 2 sequences. Among these hGRalpha splice variants, three of them, 1A1/E2dist hGRalpha, 1A2/E2prox hGRalpha, and 1A3/E3 hGRalpha, arise from the hGR 1A promoter, while 1B/E3 hGRalpha comes from the hGR 1B promoter. When fused to Flag and enhanced green fluorescent protein (EGFP) tags at the carboxy terminus, all transcript variants can be correctly translated in vitro and in vivo. The Flag-tagged hGRalpha protein variants can functionally bind to a glucocorticoid response element (GRE) and can mediate hormonal stimulation of a pGRE-luciferase reporter gene. Compared to the "classical", native hGRalpha, these four variants exhibit a cell type-specific activation of a reporter gene, and this is influenced by the hGRalpha 3' untranslated region in the hGR transcript. When equal amounts of the cDNAs for these GRalpha variant proteins are transfected into cells, they can exhibit lower or higher transcriptional activation compared to the classical GR. Furthermore, the EGFP-tagged proteins are nuclear localized, even in the absence of hormone. Using quantitative reverse transcription PCR, we found that these transcripts exist at a low level in CEM-C7 cells and IM-9 cells, although the concentrations of the 1A3/E3 hGRalpha and 1B/E3 hGRalpha transcripts are higher than for hGRbeta transcripts, while 1A1/E2dist hGRalphaand 1A2/E2prox hGRalpha transcript levels are comparable to the 1A1 hGRalpha and 1A2 hGRalpha (without the exon 2 deletions) transcript levels, respectively. Because these novel hGR, N-terminal deleted, protein variants have altered biological activity, their expression could potentially affect the hormone sensitivity or resistance in leukemia and be useful in diagnosing hormone-sensitive or -resistant disease.

Three mechanisms are involved in glucocorticoid receptor autoregulation in a human T-lymphoblast cell line.

Glucocorticoids up-regulate the glucocorticoid receptor (GR) in the human T-lymphoblast cell line CEM-C7. One mechanism for the up-regulation of the GR protein is the well-known up-regulation of GR transcripts. We have investigated the effect of other factors on the up-regulation. At least three promoters (1A, 1B, and 1C) exist, which give rise to GR transcripts with different exon 1 sequences. Transcripts with different exon 1 sequences have similar stabilities. Glucocorticoids have little, if any, effect on mRNA stability. In transfection experiments of the GR-deficient mouse fibroblast cell line E8.2, different exon 1 sequences furthermore caused no significant differences in the translational efficiencies of GR transcripts. However, the ratio between the concentrations of the glucocorticoid receptor B (GR-B) isoform and the glucocorticoid receptor A (GR-A) isoform was higher for transcripts containing the exon 1A3 sequence arising from promoter 1A than in transcripts containing exon 1 sequences from promoters 1B and 1C. Because the GR-B isoform is more active in transactivation then GR-A, this would tend to fine-tune glucocorticoid responsiveness of CEM-C7 cells, which express exon 1A3-containing transcripts. We also found that glucocorticoids do not decrease the stability of the GR protein in CEM-C7 cells. In contrast to other cell lines that downregulate GR expression in response to glucocorticoids, CEM-C7 lymphoblasts possess three mechanisms ensuring high glucocorticoid responsiveness: an up-regulation of GR mRNA by glucocorticoids, no destabilization of GR protein by glucocorticoids, and a high activity of promoter 1A with concomitant high expression of the GR-B isoform.