CDK5RAP3 is a co-factor for the oncogenic transcription factor STAT3.

The transcription factor STAT3 regulates genes governing critical cellular processes such as proliferation, survival, and self-renewal. While STAT3 transcriptional function is activated rapidly and transiently in response to physiologic signals, through a variety of mechanisms it can become constitutively activated in the pathogenesis of cancer. This leads to chronic expression of genes that underlie malignant cellular behavior. However, STAT3 is known to interact with other proteins, which may modulate its function. Understanding these interactions can provide insights into novel aspects of STAT3 function and may also suggest strategies to therapeutically target the large number of cancers driven by constitutively activated STAT3. To identify critical modulators of STAT3 transcriptional function, we performed an RNA-interference based screen in a cell-based system that allows quantitative measurement of STAT3 activity. From this approach, we identified CDK5 kinase regulatory-subunit associated protein 3 (CDK5RAP3) as an enhancer of STAT3-dependent gene expression. We found that STAT3 transcriptional function is modulated by CDK5RAP3 in cancer cells, and silencing CDK5RAP3 reduces STAT3-mediated tumorigenic phenotypes including clonogenesis and migration. Mechanistically, CDK5RAP3 binds to STAT3-regulated genomic loci, in a STAT3-dependent manner. In primary human breast cancers, the expression of CDK5RAP3 expression was associated with STAT3 gene expression signatures as well as the expression of individual STAT3 target genes. These findings reveal a novel aspect of STAT3 transcriptional function and potentially provide both a biomarker of enhanced STAT3-dependent gene expression as well as a unique mechanism to therapeutically target STAT3.

Targeting STAT5 in hematologic malignancies through inhibition of the bromodomain and extra-terminal (BET) bromodomain protein BRD2.

The transcription factor signal STAT5 is constitutively activated in a wide range of leukemias and lymphomas, and drives the expression of genes necessary for proliferation, survival, and self-renewal. Thus, targeting STAT5 is an appealing therapeutic strategy for hematologic malignancies. Given the importance of bromodomain-containing proteins in transcriptional regulation, we considered the hypothesis that a pharmacologic bromodomain inhibitor could inhibit STAT5-dependent gene expression. We found that the small-molecule bromodomain and extra-terminal (BET) bromodomain inhibitor JQ1 decreases STAT5-dependent (but not STAT3-dependent) transcription of both heterologous reporter genes and endogenous STAT5 target genes. JQ1 reduces STAT5 function in leukemia and lymphoma cells with constitutive STAT5 activation, or inducibly activated by cytokine stimulation. Among the BET bromodomain subfamily of proteins, it seems that BRD2 is the critical mediator for STAT5 activity. In experimental models of acute T-cell lymphoblastic leukemias, where activated STAT5 contributes to leukemia cell survival, Brd2 knockdown or JQ1 treatment shows strong synergy with tyrosine kinase inhibitors (TKI) in inducing apoptosis in leukemia cells. In contrast, mononuclear cells isolated form umbilical cord blood, which is enriched in normal hematopoietic precursor cells, were unaffected by these combinations. These findings indicate a unique functional association between BRD2 and STAT5, and suggest that combinations of JQ1 and TKIs may be an important rational strategy for treating leukemias and lymphomas driven by constitutive STAT5 activation.

STAT5 outcompetes STAT3 to regulate the expression of the oncogenic transcriptional modulator BCL6.

Inappropriate activation of the transcription factors STAT3 and STAT5 has been shown to drive cancer pathogenesis through dysregulation of genes involved in cell survival, growth, and differentiation. Although STAT3 and STAT5 are structurally related, they can have opposite effects on key genes, including BCL6. BCL6, a transcriptional repressor, has been shown to be oncogenic in diffuse large B cell lymphoma. BCL6 also plays an important role in breast cancer pathogenesis, a disease in which STAT3 and STAT5 can be activated individually or concomitantly. To determine the mechanism by which these oncogenic transcription factors regulate BCL6 transcription, we analyzed their effects at the levels of chromatin and gene expression. We found that STAT3 increases expression of BCL6 and enhances recruitment of RNA polymerase II phosphorylated at a site associated with transcriptional initiation. STAT5, in contrast, represses BCL6 expression below basal levels and decreases the association of RNA polymerase II at the gene. Furthermore, the repression mediated by STAT5 is dominant over STAT3-mediated induction. STAT5 exerts this effect by displacing STAT3 from one of the two regulatory regions to which it binds. These findings may underlie the divergent biology of breast cancers containing activated STAT3 alone or in conjunction with activated STAT5.

The STAT5 Inhibitor Pimozide Displays Efficacy in Models of Acute Myelogenous Leukemia Driven by FLT3 Mutations.

Activation of the transcription factor STAT5 is essential for the pathogenesis of acute myelogenous leukemia (AML) containing the FLT3 internal tandem duplication (ITD) mutation. FLT3 ITD is a constitutively active tyrosine kinase that drives the activation of STAT5, leading to the growth and survival of AML cells. Although there has been some success in identifying tyrosine kinase inhibitors that block the function of FLT3 ITD, there remains a continued need for effective treatment of this disease. We have identified the psychotropic drug pimozide as an effective inhibitor of STAT5 function. Pimozide inhibits the tyrosine phosphorylation of STAT5, leading to the death of AML cells through the induction of apoptosis. Pimozide shows a combinatorial effect with the tyrosine kinase inhibitors midostaurin (PKC412) and sunitinib in the inhibition of STAT5 tyrosine phosphorylation and the induction of apoptosis. Significantly, pimozide reduces the tumor burden in a mouse model of FLT3-driven AML. Therefore, identifying STAT5 inhibitors may provide a new avenue for the treatment of AML, and these may be effective alone or in combination with tyrosine kinase inhibitors.

STAT signaling in the pathogenesis and treatment of myeloid malignancies.

STAT transcription factors play a critical role in mediating the effects of cytokines on myeloid cells. As STAT target genes control key processes such as survival, proliferation and self-renewal, it is not surprising that constitutive activation of STATs, particularly STAT3 and STAT5, are common events in many myeloid tumors. STATs are activated both by mutant tyrosine kinases as well as other pathogenic events, and continued activation of STATs is common in the setting of resistance to kinase inhibitors. Thus, the targeting of STATs, alone or in combination with other drugs, will likely have increasing importance for cancer therapy.

Pyrimethamine inhibits adult polycystic kidney disease by modulating STAT signaling pathways.

Autosomal dominant polycystic kidney disease (ADPKD) is a commonly inherited disorder mostly caused by mutations in PKD1, encoding polycystin-1 (PC1). The disease is characterized by development and growth of epithelium-lined cyst in both kidneys, often leading to renal failure. There is no specific treatment for this disease. Here, we report a sustained activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) in ischemic injured and uninjured Pkd1 knockout polycystic kidneys and in human ADPKD kidneys. Through a chemical library screen, we identified the anti-parasitic compound pyrimethamine as an inhibitor of STAT3 function. Treatment with pyrimethamine decreases cell proliferation in human ADPKD cells and blocks renal cyst formation in an adult and a neonatal PKD mouse model. Moreover, we demonstrated that a specific STAT3 inhibitor, S3I-201, reduces cyst formation and growth in a neonatal PKD mouse model. Our results suggest that PC1 acts as a negative regulator of STAT3 and that blocking STAT3 signaling with pyrimethamine or similar drugs may be an attractive therapy for human ADPKD.

A chemical biology approach to developing STAT inhibitors: molecular strategies for accelerating clinical translation.

STAT transcription factors transduce signals from the cell surface to the nucleus, where they regulate the expression of genes that control proliferation, survival, self-renewal, and other critical cellular functions. Under normal physiological conditions, the activation of STATs is tightly regulated. In cancer, by contrast, STAT proteins, particularly STAT3 and STAT5, become activated constitutively, thereby driving the malignant phenotype of cancer cells. Since these proteins are largely dispensable in the function of normal adult cells, STATs represent a potentially important target for cancer therapy. Although transcription factors have traditionally been viewed as suboptimal targets for pharmacological inhibition, chemical biology approaches have been particularly fruitful in identifying compounds that can modulate this pathway through a variety of mechanisms. STAT inhibitors have notable anti-cancer effects in many tumor systems, show synergy with other therapeutic modalities, and have the potential to eradicate tumor stem cells. Furthermore, STAT inhibitors identified through the screening of chemical libraries can then be employed in large scale analyses such as gene expression profiling, RNA interference screens, or large-scale tumor cell line profiling. Data derived from these studies can then provide key insights into mechanisms of STAT signal transduction, as well as inform the rational design of targeted therapeutic strategies for cancer patients.

RON (MST1R) is a novel prognostic marker and therapeutic target for gastroesophageal adenocarcinoma.

RON (MST1R) is one of two members of the MET receptor tyrosine kinase family, along with parent receptor MET. RON has a putative role in several cancers, but its expression and function is poorly characterized in gastroesophageal adenocarcinoma. A recognized functional role of MET tyrosine kinase in gastroesophageal cancer has led to early phase clinical trials using MET inhibitors, with unimpressive results. Therefore, the role of RON in gastroesophageal cancer, as well as its role in cooperative signaling with MET and as a mechanism of resistance to MET inhibition, was studied in gastroesophageal tissues and cell lines. By IHC, RON was highly over-expressed in 74% of gastroesophageal samples (n=94), and over-expression was prognostic of poor survival (p=0.008); RON and MET co-expression occurred in 43% of samples and was prognostic of worst survival (p=0.03). High MST1R gene copy number by quantitative polymerase chain reaction, and confirmed by fluorescence in situ hybridization and/or array comparative genomic hybridization, was seen in 35.5% (16/45) of cases. High MST1R gene copy number correlated with poor survival (p=0.01), and was associated with high MET and ERBB2 gene copy number. A novel somatic MST1R juxtamembrane mutation R1018G was found in 11% of samples. RON signaling was functional in cell lines, activating downstream effector STAT3, and resulted in increased viability over controls. RON and MET co-stimulation assays led to enhanced malignant phenotypes over stimulation of either receptor alone. Growth inhibition as evidenced by viability and apoptosis assays was optimal using novel blocking monoclonal antibodies to both RON and MET, versus either alone. SU11274, a classic MET small molecule tyrosine kinase inhibitor, blocked signaling of both receptors, and proved synergistic when combined with STAT3 inhibition (combination index < 1). These preclinical studies define RON as an important novel prognostic marker and therapeutic target for gastroesophageal cancer warranting further investigation.

Antimyeloma activity of a multitargeted kinase inhibitor, AT9283, via potent Aurora kinase and STAT3 inhibition either alone or in combination with lenalidomide.

PURPOSE: Aurora kinases, whose expression is linked to genetic instability and cellular proliferation, are being investigated as novel therapeutic targets in multiple myeloma (MM). In this study, we investigated the preclinical activity of a small-molecule multitargeted kinase inhibitor, AT9283, with potent activity against Aurora kinase A, Aurora kinase B, and Janus kinase 2/3. EXPERIMENTAL DESIGN: We evaluated the in vitro antimyeloma activity of AT9283 alone and in combination with lenalidomide and the in vivo efficacy by using a xenograft mouse model of human MM. RESULTS: Our data showed that AT9283 induced cell-growth inhibition and apoptosis in MM. Studying the apoptosis mechanism of AT9283 in MM, we observed features consistent with both Aurora kinase A and Aurora kinase B inhibition, such as increase of cells with polyploid DNA content, decrease in phospho-histone H3, and decrease in phospho-Aurora A. Importantly, AT9283 also inhibited STAT3 tyrosine phosphorylation in MM cells. Genetic depletion of STAT3, Aurora kinase A, or Aurora kinase B showed growth inhibition of MM cells, suggesting a role of AT9283-induced inhibition of these molecules in the underlying mechanism of MM cell death. In vivo studies showed decreased MM cell growth and prolonged survival in AT9283-treated mice compared with controls. Importantly, combination studies of AT9283 with lenalidomide showed significant synergistic cytotoxicity in MM cells, even in the presence of bone marrow stromal cells. Enhanced cytotoxicity was associated with increased inhibition of phosphorylated STAT3 and phosphorylated extracellular signal-regulated kinase. CONCLUSIONS: Demonstration of in vitro and in vivo anti-MM activity of AT9283 provides the rationale for the clinical evaluation of AT9283 as monotherapy and in combination therapy for treating patients with MM.

The STAT5 inhibitor pimozide decreases survival of chronic myelogenous leukemia cells resistant to kinase inhibitors.

The transcription factor STAT5 is an essential mediator of the pathogenesis of chronic myelogenous leukemia (CML). In CML, the BCR/ABL fusion kinase causes the constitutive activation of STAT5, thereby driving the expression of genes promoting survival. BCR/ABL kinase inhibitors have become the mainstay of therapy for CML, although CML cells can develop resistance through mutations in BCR/ABL. To overcome this problem, we used a cell-based screen to identify drugs that inhibit STAT-dependent gene expression. Using this approach, we identified the psychotropic drug pimozide as a STAT5 inhibitor. Pimozide decreases STAT5 tyrosine phosphorylation, although it does not inhibit BCR/ABL or other tyrosine kinases. Furthermore, pimozide decreases the expression of STAT5 target genes and induces cell cycle arrest and apoptosis in CML cell lines. Pimozide also selectively inhibits colony formation of CD34(+) bone marrow cells from CML patients. Importantly, pimozide induces similar effects in the presence of the T315I BCR/ABL mutation that renders the kinase resistant to presently available inhibitors. Simultaneously inhibiting STAT5 with pimozide and the kinase inhibitors imatinib or nilotinib shows enhanced effects in inhibiting STAT5 phosphorylation and in inducing apoptosis. Thus, targeting STAT5 may be an effective strategy for the treatment of CML and other myeloproliferative diseases.

Discovery and characterization of novel mutant FLT3 kinase inhibitors.

For a subpopulation of acute myeloid leukemia (AML) patients, the constitutively activated tyrosine kinase, mutant FLT3, has emerged as a promising target for therapy. The development of drug resistance, however, is a growing concern for mutant FLT3 inhibitors, such as PKC412. Potential therapeutic benefit can arise from the combination of two structurally diverse inhibitors that target-but bind differently to-the same protein or from two inhibitors with completely different mechanisms of action. Thus, there is a need for identification and development of novel FLT3 inhibitors that have the ability to positively combine with PKC412 or standard chemotherapeutic agents used to treat AML as a way to suppress the development of drug resistance and consequently prolong disease remission. Here, we report the effects of the novel type II ATP-competitive inhibitors, HG-7-85-01 and HG-7-86-01, which potently and selectively target mutant FLT3 protein kinase activity and inhibit the proliferation of cells harboring FLT3-ITD or FLT3 kinase domain point mutants via induction of apoptosis and cell cycle inhibition. Antileukemic activity of HG-7-85-01 was shown in vivo to be comparable with that observed with PKC412 in a bioluminescence assay using NCr nude mice harboring Ba/F3-FLT3-ITD-luc+ cells. HG-7-85-01 was also observed to override PKC412 resistance. Finally, HG-7-85-01 and HG-7-86-01 synergized with PKC412 and standard chemotherapeutic agents against mutant PKC412-sensitive and some PKC412-resistant, FLT3-positive cells. Thus, we present a structurally novel class of FLT3 inhibitors that warrants consideration for clinical testing against drug-resistant disease in AML patients.

Microtubule-targeted chemotherapeutic agents inhibit signal transducer and activator of transcription 3 (STAT3) signaling.

The transcription factor signal transducer and activator of transcription 3 (STAT3) is inappropriately activated in the majority of breast tumors, especially in aggressive and invasive ones. In addition to driving the expression of genes promoting malignancy, STAT3 associates with tubulin and can promote cell migration. Because microtubule-targeted drugs are among the most active agents used in the treatment of breast cancer, we examined whether microtubule-based chemotherapy modulates STAT3 activity. When treated with paclitaxel or vinorelbine, breast cancer cells with constitutive activation of STAT3 display a loss of STAT3 phosphorylation, and paclitaxel disrupts the interaction of STAT3 with tubulin. Paclitaxel also inhibits cytokine-induced STAT3 activation. This effect is specific for microtubule-targeted agents, because other chemotherapeutic drugs, such as doxorubicin, have no effect on STAT3. The loss of STAT3 tyrosine phosphorylation is also reflected in an inhibition of expression of STAT3 target genes. This effect is not restricted to breast cancer, because similar effects are also seen in ovarian cancer and prostate cancer cells. Thus, in addition to their role in disrupting microtubule function, microtubule-targeted agents also suppress STAT3 signaling. This may be an important component of their activity, raising the possibility that microtubule targeted therapy may be particularly effective in tumors characterized by STAT3 activation.

Reciprocal effects of STAT5 and STAT3 in breast cancer.

Breast cancer is often associated with inappropriate activation of transcription factors involved in normal mammary development. Two related transcription factors, signal transducer and activator of transcription (STAT) 5 and STAT3, play important and distinct roles in mammary development and both can be activated in breast cancer. However, the relative contribution of these STATs to mammary tumorigenesis is unknown. We have found that primary human breast tumors displaying activation of both STATs are more differentiated than those with STAT3 activation alone and display more favorable prognostic characteristics. To understand this difference, we have analyzed the effect of these STATs on gene regulation and phenotype of mammary carcinoma cells. STAT5 and STAT3 mediate opposing effects on several key target genes, with STAT5 exerting a dominant role. Using a model system of paired breast cancer cell lines, we found that coactivation of STAT5 and STAT3 leads to decreased proliferation and increased sensitivity to the chemotherapeutic drugs paclitaxel and vinorelbine compared with cells that have only STAT3 activation. Thus, STAT5 can modify the effects of STAT3 from the level of gene expression to cellular phenotype and analysis of the activation state of both STAT5 and STAT3 may provide important diagnostic and prognostic information in breast cancer.

Nifuroxazide inhibits survival of multiple myeloma cells by directly inhibiting STAT3.

Constitutive activation of the transcription factor STAT3 contributes to the pathogenesis of many cancers, including multiple myeloma (MM). Since STAT3 is dispensable in most normal tissue, targeted inhibition of STAT3 is an attractive therapy for patients with these cancers. To identify STAT3 inhibitors, we developed a transcriptionally based assay and screened a library of compounds known to be safe in humans. We found the drug nifuroxazide to be an effective inhibitor of STAT3 function. Nifuroxazide inhibits the constitutive phosphorylation of STAT3 in MM cells by reducing Jak kinase autophosphorylation, and leads to down-regulation of the STAT3 target gene Mcl-1. Nifuroxazide causes a decrease in viability of primary myeloma cells and myeloma cell lines containing STAT3 activation, but not normal peripheral blood mononuclear cells. Although bone marrow stromal cells provide survival signals to myeloma cells, nifuroxazide can overcome this survival advantage. Reflecting the interaction of STAT3 with other cellular pathways, nifuroxazide shows enhanced cytotoxicity when combined with either the histone deacetylase inhibitor depsipeptide or the MEK inhibitor UO126. Therefore, using a mechanistic-based screen, we identified the clinically relevant drug nifuroxazide as a potent inhibitor of STAT signaling that shows cytotoxicity against myeloma cells that depend on STAT3 for survival.

Identification of human STAT5-dependent gene regulatory elements based on interspecies homology.

STAT5 is a transcription factor essential for hematopoietic physiology. STAT5 functions to transduce signals from cytokines to the nucleus where it regulates gene expression. Although several important transcriptional targets of STAT5 are known, most remain unidentified. To identify novel STAT5 targets, we searched chromosomes 21 and 22 for clusters of STAT5 binding sites contained within regions of interspecies homology. We identified four such regions, including one with tandem STAT5 binding sites in the first intron of the NCAM2 gene. Unlike known STAT5 binding sites, this site is found within a very large intron and resides approximately 200 kb from the first coding exon of NCAM2. We demonstrate that this region confers STAT5-dependent transcriptional activity. We show that STAT5 binds in vivo to the NCAM2 intron in the NKL natural killer cell line and that this binding is induced by cytokines that activate STAT5. Neither STAT1 nor STAT3 bind to this region, despite sharing a consensus binding sequence with STAT5. Activation of STAT4 and STAT5 causes the accumulation of both of these STATs to the NCAM2 regulatory region. Therefore, using an informatics based approach to identify STAT5 targets, we have identified NCAM2 as both a STAT4- and STAT5-regulated gene, and we show that its expression is regulated by cytokines essential for natural killer cell survival and differentiation. This strategy may be an effective way to identify functional binding regions for transcription factors with known cognate binding sites anywhere in the genome.

IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo.

IL-2 plays a critical role in the maintenance of CD4+CD25+ FOXP3(+) regulatory T cells (Tregs) in vivo. We examined the effects of IL-2 signaling in human Tregs. In vitro, IL-2 selectively up-regulated the expression of FOXP3 in purified CD4+CD25+ T cells but not in CD4+CD25- cells. This regulation involved the binding of STAT3 and STAT5 proteins to a highly conserved STAT-binding site located in the first intron of the FOXP3 gene. We also examined the effects of low-dose IL-2 treatment in 12 patients with metastatic cancer and 9 patients with chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation. Overall, IL-2 treatment resulted in a 1.9 median fold increase in the frequency of CD4+CD25+ cells in peripheral blood as well as a 9.7 median fold increase in FOXP3 expression in CD3+ T cells. CD56+CD3- natural killer (NK) cells also expanded during IL-2 therapy but did not express FOXP3. In vitro treatment of NK cells with 5-aza-2'-deoxycytidine restored the IL-2 signaling pathway leading to FOXP3 expression, suggesting that this gene was constitutively repressed by DNA methylation in these cells. Our findings support the clinical evaluation of low-dose IL-2 to selectively modulate CD4+CD25+ Tregs and increase expression of FOXP3 in vivo.

Isolation of unique STAT5 targets by chromatin immunoprecipitation-based gene identification.

STAT5a and STAT5b are two highly related transcription factors that control essential cellular functions. Several STAT5 targets are known, although it is likely that most remain uncharacterized. To identify a more complete set of STAT5-regulated genes, we used a modification of the chromatin immunoprecipitation procedure, which does not presuppose any information regarding these targets. Employing Ba/f3 cells in which STAT5 is activated by interleukin-3, we have identified novel STAT5 binding sites that may be regulatory regions for nearby genes. These sites are typically found far from transcription start sites, and most do not contain CpG islands, indicating that they are not in traditional promoter regions. Nonetheless, when the expression of genes near these STAT5 binding sites was examined, all were expressed in Ba/f3 cells, and most were modulated by interleukin-3. Furthermore, genes identified by this strategy show unique expression patterns in acute leukemias, tumors characterized by activated STAT5. Whereas both STAT5 isoforms bound to all promoters tested, STAT5a and STAT5b bound with different kinetics, suggesting that at least some of the differences between the functions of these two proteins are mediated by their DNA binding activity. Therefore, this method of transcription factor target identification represents an effective strategy to isolate transcription factor targets in an unbiased fashion, and it has revealed many novel STAT5-dependent regulatory regions outside of traditional promoters.

The amino terminal and E2F interaction domains are critical for C/EBP alpha-mediated induction of granulopoietic development of hematopoietic cells.

The transcription factor C/EBP alpha (CCAAT/enhancer binding protein alpha) is critical for granulopoiesis. Gene disruption in mice blocks early granulocyte differentiation and disruption of C/EBP alpha function has been implicated in human acute myeloid leukemia (AML), but no systematic structure-function analysis has been undertaken to identify the mechanisms involved in C/EBP alpha-mediated granulocyte differentiation. Here we demonstrate that loss of either of 2 key regions results in disruption of C/EBP alpha granulocytic development: the amino terminus and specific residues residing on the non-DNA binding face of the basic region. Mutation of either results in loss of C/EBP alpha inhibition of E2F and down-regulation of c-Myc, but only mutation of the basic region results in loss of physical interaction with E2F. In contrast, while the amino terminal mutant retains the ability to interact with E2F, this mutant fails to bind a C/EBP alpha site efficiently, fails to activate C/EBP alpha target genes, and is also defective in inhibition of E2F activity. These results further emphasize the importance of inhibition of proliferative pathways in granulopoiesis and demonstrate that several regions of the C/EBP alpha protein are involved in this mechanism.