Novel FOXM1 inhibitor STL001 sensitizes human cancers to a broad-spectrum of cancer therapies.

Forkhead box protein M1 (FOXM1) is often overexpressed in human cancers and strongly associated with therapy resistance and less good patient survival. The chemotherapy options for patients with the most aggressive types of solid cancers remain very limited because of the acquired drug resistance, making the therapy less effective. NPM1 mutation through the inactivation of FOXM1 via FOXM1 relocalization to the cytoplasm confers more favorable treatment outcomes for AML patients, confirming FOXM1 as a crucial target to overcome drug resistance. Pharmacological inhibition of FOXM1 could be a promising approach to sensitize therapy-resistant cancers. Here, we explore a novel FOXM1 inhibitor STL001, a first-generation modification drug of our previously reported FOXM1 inhibitor STL427944. STL001 preserves the mode of action of the STL427944; however, STL001 is up to 50 times more efficient in reducing FOXM1 activity in a variety of solid cancers. The most conventional cancer therapies studied here induce FOXM1 overexpression in solid cancers. The therapy-induced FOXM1 overexpression may explain the failure or reduced efficacy of these drugs in cancer patients. Interestingly, STL001 increased the sensitivity of cancer cells to conventional cancer therapies by suppressing both the high-endogenous and drug-induced FOXM1. Notably, STL001 does not provide further sensitization to FOXM1-KD cancer cells, suggesting that the sensitization effect is conveyed specifically through FOXM1 suppression. RNA-seq and gene set enrichment studies revealed prominent suppression of FOXM1-dependent pathways and gene ontologies. Also, gene regulation by STL001 showed extensive overlap with FOXM1-KD, suggesting a high selectivity of STL001 toward the FOXM1 regulatory network. A completely new activity of FOXM1, mediated through steroid/cholesterol biosynthetic process and protein secretion in cancer cells was also detected. Collectively, STL001 offers intriguing translational opportunities as combination therapies targeting FOXM1 activity in a variety of human cancers driven by FOXM1.

Effect of the LSD1 inhibitor RN-1 on γ-globin and global gene expression during erythroid differentiation in baboons (Papio anubis).

Elevated levels of Fetal Hemoglobin interfere with polymerization of sickle hemoglobin thereby reducing anemia, lessening the severity of symptoms, and increasing life span of patients with sickle cell disease. An affordable, small molecule drug that stimulates HbF expression in vivo would be ideally suited to treat the large numbers of SCD patients that exist worldwide. Our previous work showed that administration of the LSD1 (KDM1A) inhibitor RN-1 to normal baboons increased Fetal Hemoglobin (HbF) and was tolerated over a prolonged treatment period. HbF elevations were associated with changes in epigenetic modifications that included increased levels of H3K4 di-and tri-methyl lysine at the γ-globin promoter. While dramatic effects of the loss of LSD1 on hematopoietic differentiation have been observed in murine LSD1 gene deletion and silencing models, the effect of pharmacological inhibition of LSD1 in vivo on hematopoietic differentiation is unknown. The goal of these experiments was to investigate the in vivo mechanism of action of the LSD1 inhibitor RN-1 by determining its effect on γ-globin expression in highly purified subpopulations of bone marrow erythroid cells enriched for varying stages of erythroid differentiation isolated directly from baboons treated with RN-1 and also by investigating the effect of RN1 on the global transcriptome in a highly purified population of proerythroblasts. Our results show that RN-1 administered to baboons targets an early event during erythroid differentiation responsible for γ-globin repression and increases the expression of a limited number of genes including genes involved in erythroid differentiation such as GATA2, GFi-1B, and LYN.

Anti-HIV Drugs Cause Mitochondrial Dysfunction in Monocyte-Derived Macrophages.

Combination antiretroviral therapy (cART) dramatically changed the face of the HIV/AIDS pandemic, making it one of the most prominent medical breakthroughs of the past 3 decades. However, as the life span of persons living with HIV (PLWH) continues to approach that of the general population, the same cannot be said regarding their quality of life. PLWH are affected by comorbid conditions such as high blood pressure, diabetes, and neurocognitive impairment at a higher rate and increased severity than their age-matched counterparts. PLWH also have higher levels of inflammation, the drivers of which are not entirely clear. As cART treatment is lifelong, we assessed here the effects of cART, independent of HIV, on primary human monocyte-derived macrophages (MDMs). MDMs were unskewed or skewed to an alternative phenotype and treated with Atripla or Triumeq, two first-line cART treatments. We report that Triumeq skewed alternative MDMs toward an inflammatory nonsenescent phenotype. Both Atripla and Triumeq caused mitochondrial dysfunction, specifically efavirenz and abacavir. Additionally, transcriptome sequencing (RNA-seq) demonstrated that both Atripla and Triumeq caused differential regulation of genes involved in immune regulation and cell cycle and DNA repair. Collectively, our data demonstrate that cART, independent of HIV, alters the MDM phenotype. This suggests that cART may contribute to cell dysregulation in PLWH that subsequently results in increased susceptibility to comorbidities.

FOXM1-AKT Positive Regulation Loop Provides Venetoclax Resistance in AML.

Forkhead box protein M1 (FOXM1) is a crucial regulator of cancer development and chemoresistance. It is often overexpressed in acute myeloid leukemia (AML) and is associated with poor survival and reduced efficacy of cytarabine therapy. Molecular mechanisms underlying high FOXM1 expression levels in malignant cells are still unclear. Here we demonstrate that AKT and FOXM1 constitute a positive autoregulatory loop in AML cells that sustains high activity of both pro-oncogenic regulators. Inactivation of either AKT or FOXM1 signaling results in disruption of whole loop, coordinated suppression of FOXM1 or AKT, respectively, and similar transcriptomic changes. AML cells with inhibited AKT activity or stable FOXM1 knockdown display increase in HOXA genes expression and BCL2L1 suppression that are associated with prominent sensitization to treatment with Bcl-2 inhibitor venetoclax. Taken together, our data indicate that AKT and FOXM1 in AML cells should not be evaluated as single independent regulators but as two parts of a common FOXM1-AKT positive feedback circuit. We also report for the first time that FOXM1 inactivation can overcome AML venetoclax resistance. Thus, targeting FOXM1-AKT loop may open new possibilities in overcoming AML drug resistance and improving outcomes for AML patients.

Novel FOXM1 inhibitor identified via gene network analysis induces autophagic FOXM1 degradation to overcome chemoresistance of human cancer cells.

FOXM1 transcription factor is an oncogene and a master regulator of chemoresistance in multiple cancers. Pharmacological inhibition of FOXM1 is a promising approach but has proven to be challenging. We performed a network-centric transcriptomic analysis to identify a novel compound STL427944 that selectively suppresses FOXM1 by inducing the relocalization of nuclear FOXM1 protein to the cytoplasm and promoting its subsequent degradation by autophagosomes. Human cancer cells treated with STL427944 exhibit increased sensitivity to cytotoxic effects of conventional chemotherapeutic treatments (platinum-based agents, 5-fluorouracil, and taxanes). RNA-seq analysis of STL427944-induced gene expression changes revealed prominent suppression of gene signatures characteristic for FOXM1 and its downstream targets but no significant changes in other important regulatory pathways, thereby suggesting high selectivity of STL427944 toward the FOXM1 pathway. Collectively, the novel autophagy-dependent mode of FOXM1 suppression by STL427944 validates a unique pathway to overcome tumor chemoresistance and improve the efficacy of treatment with conventional cancer drugs.

Induction of Antigen-Independent Proliferation of Regulatory T-Cells by TNF Superfamily Ligands OX40L and GITRL.

TNF receptor superfamily comprises many T-cell costimulatory receptors, including TNFRSF1, TNFRSF2, TNFRSF4 (OX40), TNFRSF9 (4-1BB), TNFRSF18 (GITR), and TNFRSF7 (CD27). Signaling through these costimulatory stimulatory receptors can promote conventional T-cell (Tconv) proliferation, and effector functions in an antigen-dependent manner. Thus, agonistic antibodies and ligands for OX40, 4-1BB, GITR, and CD27 have been tested for inducing T-cell-mediated antitumor responses in several cancers. However, recently emerging reports show critical role for TNFR signaling in regulatory T-cell (Treg) differentiation and expansion, which might suppress effector T-cell proliferation and functions. Here, we show preferential over expression of TNFR2, OX40, 4-1BB, and GITR in Treg cells over Tconv cells, and the ability of OX40L and GITRL to induce selective proliferation of Treg cells, but not Tconv cells, in an antigen-independent manner. We describe the standard protocols used for Affymetrix gene expression profiling, T-cell isolation, and Cell Trace Violet-based cell proliferation assay.

Expression profiling of genes regulated by sphingosine kinase1 signaling in a murine model of hyperoxia induced neonatal bronchopulmonary dysplasia.

BACKGROUND: Sphingosine- 1-Phosphate (S1P) is a bioactive lipid and an intracellular as well as an extracellular signaling molecule. S1P ligand specifically binds to five related cell surface G-protein-coupled receptors (S1P1-5). S1P levels are tightly regulated by its synthesis catalyzed by sphingosine kinases (SphKs) 1 & 2 and catabolism by S1P phosphatases, lipid phosphate phosphatases and S1P lyase. We previously reported that knock down of SphK1 (Sphk1 -/- ) in a neonatal mouse BPD model conferred significant protection against hyperoxia induced lung injury. To better understand the underlying molecular mechanisms, genome-wide gene expression profiling was performed on mouse lung tissue using Affymetrix MoGene 2.0 array. RESULTS: Two-way ANOVA analysis was performed and differentially expressed genes under hyperoxia were identified using Sphk1 -/- mice and their wild type (WT) equivalents. Pathway (PW) enrichment analyses identified several signaling pathways that are likely to play a key role in hyperoxia induced lung injury in the neonates. These included signaling pathways that were anticipated such as those involved in lipid signaling, cell cycle regulation, DNA damage/apoptosis, inflammation/immune response, and cell adhesion/extracellular matrix (ECM) remodeling. We noted hyperoxia induced downregulation of the expression of genes related to mitotic spindle formation in the WT which was not observed in Sphk1 -/- neonates. Our data clearly suggests a role for SphK1 in neonatal hyperoxic lung injury through elevated inflammation and apoptosis in lung tissue. Further, validation by RT-PCR on 24 differentially expressed genes showed 83% concordance both in terms of fold change and vectorial changes. Our findings are in agreement with previously reported human BPD microarray data and completely support our published in vivo findings. In addition, the data also revealed a significant role for additional unanticipitated signaling pathways involving Wnt and GADD45. CONCLUSION: Using SphK1 knockout mice and differential gene expression analysis, we have shown here that S1P/SphK1 signaling plays a key role in promoting hyperoxia induced DNA damage, inflammation, apoptosis and ECM remodeling in neonatal lungs. It also appears to suppress pro-survival cellular responses involved in normal lung development. We therefore propose SphK1 as a therapeutic target for the development drugs to combat BPD.

A differential gene expression study: Ptpn6 (SHP-1)-insufficiency leads to neutrophilic dermatosis-like disease (NDLD) in mice.

BACKGROUND: Irradiated syngeneic wild-type mice developed the same neutrophilic dermatosis-like disease (NDLD) after adoptive transfer of bone marrow cells from Ptpn6(meb2/meb2) mutant mice. OBJECTIVE: To analyze differentially expressed genes in the bone marrow of mice with NDLD to gain insight into the role of Ptpn6 in myelopoietic bone marrow pathology, and the mechanisms by which Ptpn6 insufficiency in the hematopoietic cells can lead to the development of skin lesions. METHODS: As Ptpn6 is involved in a myriad of signaling pathways, we used a global approach with microarray technology for the first time to characterize changes in the bone marrow and skin of motheaten-type mice. RESULTS: A total number of 1,511 probe sets in the bone marrow showed at least two-fold changes with FDR <0.05, of which 256 probe sets had over four-fold changes. A group of 63 genes in the bone marrow of NDLD mice had more than a 4-fold change with FDR <0.0001. From 503 genes encoding proteins with ITIM motif that binds to Ptpn6, 109 were up-regulated and 83 were down-regulated. We found that genes encoding hematopoietic receptors, neutrophil chemoattractants, Toll-like receptors (Tlr1, Tlr2 and Tlr4) and C-type lectin innate immunity receptors (Clec4e, Clec4d, Clec4n, Clec4a2 and Clec4a3) were significantly up-regulated in both NDLD bone marrow and skin. The Il1b gene was also significantly overexpressed in skin samples, confirming the importance of the IL-1/TLR pathway in the development of early skin inflammation in NDLD mice. CONCLUSION: Our results suggest that innate immunity genes play a major role in development of neutrophilic dermatosis-like disease in mice.

Correlative Analysis of miRNA Expression and Oncotype Dx Recurrence Score in Estrogen Receptor Positive Breast Carcinomas.

Altered expression of miRNAs has been observed in many types of cancer, including breast cancer, and shown to contribute to cancer growth, aggressiveness, and response to therapies. In this pilot study, we investigated the possible correlation of miRNAs with risk of recurrence of estrogen receptor positive, lymph node-negative mammary carcinomas as determined by the Oncotype DX® Breast Cancer assay. To accomplish this, we extracted RNA from a collection of breast carcinomas that had previously been analyzed by Oncotype DX®. Multiple Let-7 family members were negatively correlated with the recurrence score (RS), which is consistent with their tumor suppressor properties. Additional miRNAs were found to positively correlate with RS, including miR-377-5p, miR-633b, miR-548t and miR-3648. Pathway analysis of putative and validated targets suggests that these miRNAs may have a diverse range of functions that may contribute to tumor recurrence. Taken together, these findings provide evidence that a miRNA expression signature can be developed to aid existing methods to determine the risk of recurrence for women with estrogen receptor positive breast cancers treated with endocrine therapy.

Identification of human cell responses to benzene and benzene metabolites.

Benzene is a common air pollutant and confirmed carcinogen, especially in reference to the hematopoietic system. In the present study we analyzed cytokine/chemokine production by, and gene expression induction in, human peripheral blood mononuclear cells upon their exposure to the benzene metabolites catechol, hydroquinone, 1,2,4-benzenetriol, and p-benzoquinone. Protein profiling showed that benzene metabolites can stimulate the production of chemokines, the proinflammatory cytokines TNF-alpha and IL-6, and the Th2 cytokines IL-4 and IL-5. Activated cells showed concurrent suppression of anti-inflammatory cytokine IL-10 expression. We also identified changes in global gene expression patterns in response to benzene metabolite challenges by using high-density oligonucleotide microarrays. Treatment with 1,2,4-benzenetriol resulted in the suppression of genes related to the regulation of protein expression and a concomitant activation of genes that encode heat shock proteins and cytochrome P450 family members. Protein and gene expression profiling identified unique human cellular responses upon exposure to benzene and benzene metabolites.

Silvestrol regulates G2/M checkpoint genes independent of p53 activity.

As previously reported, silvestrol, a rocaglate derivative isolated from Aglaia foveolata, has similar potency to paclitaxel and camptothecin against cultured human cancer cells. Furthermore, silvestrol can inhibit cancer cell growth in mice without noticeable toxicity when administered up to 5 mg/kg body weight (the highest dose tested). The purpose of the current study was to evaluate the mechanism of silvestrol's cytotoxicity in human prostate cancer cells (LNCaP). The molecular signature induced in LNCaP cells by silvestrol was evaluated using microarray analysis. The results revealed that 20 apoptosis and cell cycle related genes were significantly altered in LNCaP cells exposed to silvestrol. These included UBL-3, p21 and p300, which were up-regulated, and p53, which was down-regulated. Since p53 expression is governed primarily at the level of translation, p53 was also evaluated by Western blot. Silvestrol caused a dose-dependent decrease in p53 protein within 30 min of exposure with no p53 detectable after 6 h. Down-regulation of p53 by silvestrol was associated with down-regulation of MDM2 and not prevented by lactacystin suggesting that silvestrol-induced degradation of p53 is not mediated by the proteasome. A slight decrease in cyclin B was observed within 6 h of silvestrol exposure and phosphatase Cdc25C protein, which activates Cdc2, was also decreased. These data demonstrate that cytotoxicity induced by silvestrol in LNCaP cells is associated with a block in the cell cycle at the G2/M checkpoint and alterations in the expression of genes regulating apoptosis and cell cycle in a manner independent of p53.

Tumor cell plasticity in uveal melanoma: microenvironment directed dampening of the invasive and metastatic genotype and phenotype accompanies the generation of vasculogenic mimicry patterns.

The histological detection of laminin-rich vasculogenic mimicry patterns in human primary uveal melanomas is associated with death from metastases. We therefore hypothesized that highly invasive uveal melanoma cells forming vasculogenic mimicry patterns after exposure to a laminin-rich three-dimensional microenvironment would differentially express genes associated with invasive and metastatic behavior. However, we discovered that genes associated with differentiation (GDF15 and ATF3) and suppression of proliferation (CDKNa1/p21) were up-regulated in highly invasive uveal melanoma cells forming vasculogenic mimicry patterns, and genes associated with promotion of invasive and metastatic behavior such as CD44, CCNE2 (cyclin E2), THBS1 (thrombospondin 1), and CSPG2 (chondroitin sulfate proteoglycan; versican) were down-regulated. After forming vasculogenic mimicry patterns, uveal melanoma cells invaded only short distances, failed to replicate, and changed morphologically from the invasive epithelioid to the indolent spindle A phenotype. In human tissue samples, uveal melanoma cells within vasculogenic mimicry patterns assumed the spindle A morphology, and the expression of Ki67 was significantly reduced in adjacent melanoma cells. Thus, the generation of vasculogenic mimicry patterns is accompanied by dampening of the invasive and metastatic uveal melanoma genotype and phenotype and underscores the plasticity of these cells in response to cues from the microenvironment.

Paracrine-stimulated gene expression profile favors estradiol production in breast tumors.

Paracrine interactions between adipose fibroblasts and malignant epithelial cells are essential for structural and hormonal support of breast tumors. Factors derived from malignant epithelial cells inhibit adipogenic differentiation of fibroblasts and upregulate expression of aromatase, which stimulates estrogen synthesis and creates a localized, growth-stimulatory environment. Here, we characterized the gene expression profile of breast adipose fibroblasts in an in vitro model of malignancy to identify other paracrine interactions that support tumor growth. Primary breast adipose fibroblasts from cancer-free women were treated with conditioned media from malignant breast epithelial cells or normal breast epithelial cells, and differences in gene expression were identified by microarray. A total of 79 differentially regulated genes encoding cytokines, enzymes, angiogenic factors, cytoskeletal proteins, extra-cellular matrix remodeling proteins, signal transduction proteins and cell surface receptors were identified, and 6 of these were verified by real-time PCR. Among these, the expression of aldo-keto reductase family 1, member C3 (AKR1C3) was upregulated. AKR1C3 has multiple enzymatic properties, including conversion of estrone to estradiol and androstenedione to testosterone. Immunoreactive AKR1C3 was detected in epithelial and stromal components of benign lesions and ductal carcinomas in situ, and in 59.8% of epithelial and 69.6% of stromal cells in invasive breast carcinomas. AKR1C3 expression was significantly higher in myoepithelial cells surrounding the neoplastic epithelium of ductal carcinoma in situ compared with those surrounding benign epithelial lesions. Importantly, AKR1C3 and aromatase mRNA levels correlated positively in 61 malignant breast tumors (R=0.3967, p=0.00156). Malignant epithelial cell-conditioned medium significantly increased formation of testosterone and estradiol from androstenedione in breast adipose fibroblasts. In conclusion, malignant epithelial cell-derived factors significantly upregulate the enzymes AKR1C3 and aromatase that catalyze a series of complementary reactions to convert the circulating precursor androstenedione to biologically active estradiol in vitro in the stromal fibroblasts, and in vivo, in stromal component of breast tumors.

Chromatin organization measured by AluI restriction enzyme changes with malignancy and is regulated by the extracellular matrix and the cytoskeleton.

Given that expression of many genes changes when cells become malignant or are placed in different microenvironments, we asked whether these changes were accompanied by global reorganization of chromatin. We reasoned that sequestration or exposure of chromatin-sensitive sites to restriction enzymes could be used to detect this reorganization. We found that AluI-sensitive sites of nonmalignant cells were relatively more exposed compared to their malignant counterparts in cultured cells and human tumor samples. Changes in exposure and sequestration of AluI-sensitive sites in normal fibroblasts versus fibrosarcoma or those transfected with oncogenes, nonmalignant breast cells versus carcinomas and poorly metastatic versus highly invasive melanoma were shown to be independent of the cell cycle and may be influenced by proteins rich in disulfide bonds. Remarkably, regardless of degree of malignancy, AluI-sensitive sites became profoundly sequestered when cells were incubated with laminin, Matrigel, or a circular RGD peptide (RGD-C), but became exposed when cells were placed on collagen I or in serum-containing medium. Disruption of the actin cytoskeleton led to exposure, whereas disruption of microtubules or intermediate filaments exerted a sequestering effect. Thus, AluI-sensitive sites are more sequestered with increasing malignant behavior, but the sequestration and exposure of these sites is exquisitely sensitive to information conferred to the cell by molecules and biomechanical forces that regulate cellular and tissue architecture.