p53-Dependent Transcriptional Responses to Interleukin-3 Signaling

p53 is critical in the normal response to a variety of cellular stresses including DNA damage and loss of p53 function is a common feature of many cancers. In hematological malignancies, p53 deletion is less common than in solid malignancies but is associated with poor prognosis and resistance to chemotherapy. Compared to their wild-type (WT) counterparts, hematopoietic progenitor cells lacking p53 have a greater propensity to survive cytokine loss, in part, due to the failure to transcribe Puma, a proapoptotic Bcl-2 family member. Using expression arrays, we have further characterized the differences that distinguish p53−/− cells from WT myeloid cells in the presence of Interleukin-3 (IL-3) to determine if such differences contribute to the increased clonogenicity and survival responses observed in p53−/− cells. We show that p53−/− cells have a deregulated intracellular signaling environment and display a more rapid and sustained response to IL-3. This was accompanied by an increase in active ERK1/2 and a dependence on an intact MAP kinase signaling pathway. Contrastingly, we find that p53−/− cells are independent on AKT for their survival. Thus, loss of p53 in myeloid cells results in an altered transcriptional and kinase signaling environment that favors enhanced cytokine signaling

The oncogenic properties of EWS/WT1 of desmoplastic small round cell tumors are unmasked by loss of p53 in murine embryonic fibroblasts

BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is characterized by the presence of a fusion protein EWS/WT1, arising from the t (11;22) (p13;q12) translocation. Here we examine the oncogenic properties of two splice variants of EWS/WT1, EWS/WT1-KTS and EWS/WT1 + KTS. METHODS: We over-expressed both EWS/WT1 variants in murine embryonic fibroblasts (MEFs) of wild-type, p53+/- and p53-/- backgrounds and measured effects on cell-proliferation, anchorage-independent growth, clonogenicity after serum withdrawal, and sensitivity to cytotoxic drugs and gamma irradiation in comparison to control cells. We examined gene expression profiles in cells expressing EWS/WT1. Finally we validated our key findings in a small series of DSRCT. RESULTS: Neither isoform of EWS/WT1 was sufficient to transform wild-type MEFs however the oncogenic potential of both was unmasked by p53 loss. Expression of EWS/WT1 in MEFs lacking at least one allele of p53 enhanced cell-proliferation, clonogenic survival and anchorage-independent growth. EWS/WT1 expression in wild-type MEFs conferred resistance to cell-cycle arrest after irradiation and daunorubicin induced apoptosis. We show DSRCT commonly have nuclear localization of p53, and copy-number amplification of MDM2/MDMX. Expression of either isoform of EWS/WT1 induced characteristic mRNA expression profiles. Gene-set enrichment analysis demonstrated enrichment of WNT pathway signatures in MEFs expressing EWS/WT1 + KTS. Wnt-activation was validated in cell lines with over-expression of EWS/WT1 and in DSRCT. CONCLUSION: In conclusion, we show both isoforms of EWS/WT1 have oncogenic potential in MEFs with loss of p53. In addition we provide the first link between EWS/WT1 and Wnt-pathway signaling. These data provide novel insights into the function of the EWS/WT1 fusion protein which characterize DSRCT

Pathway analysis of WT and <i>p53^−/−</i> samples cultured in the presence of cytokine.

<p>(A) List of activated and inactivated pathways identified by a Signaling Pathway Impact Analysis (SPIA) of array results from WT and <i>p53^−/−</i> samples. The ID is the KEGG ID, pSize indicates the number of genes in the KEGG pathway, NDE is the number of differentially expressed genes found within the pathway and pGFdr is the False Discovery pathways (FDR<0.1). Significant pathways are shown. (B) The differentially expressed genes that account for the significant SPIA pathways (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031428#pone-0031428-g002" target="_blank">Figure 2A</a>) are depicted by the heatmap. To the right of the heatmap, the dots indicate the pathways to which each gene contributes and whether a gene is represented by several of the pathways.</p

Analysis of the IL-3 signaling pathway in WT and <i>p53^−/−</i> FDM cells.

<p>(A) Independently generated WT and <i>p53^−/−</i> FDM cell lines (n = number of cell lines) were cultured in the indicated concentrations of IL-3 for 72 hours. Viability was determined using Propidium iodide (PI) exclusion detected by flow cytometry. Results show the mean +/− SEM of 3 independent experiments. (B) WT and <i>p53^−/−</i> independent FDM cell lines (n = number of cell lines) were cultured in the indicated concentrations of IL-3 doses for 72 hours and then plated in soft agar with abundant IL-3. The number of colonies was counted after 14 days and the clonogenicity (relative to the number of colonies generated in 500 pg/ml IL-3) calculated. Results show the mean +/− SEM of 2 independent experiments. (C) Lysates were generated from WT pr <i>p53^−/−</i> FDM cells following stimulation with the indicated concentrations of IL-3 after 16 hours of IL-3 deprivation. Lysates were resolved by SDS-PAGE and immunoblotted with antibodies specific to the indicated proteins. (D) Independent WT and <i>p53^−/−</i> FDM cell lines (n = number of cell lines) were treated for 24 h with an AKT inhibitor (AKTi) in the presence or absence of IL-3. Viability was determined by flow cytometric analysis of PI exclusion. Results show the mean +/− SEM of 2 independent experiments. (E) Independent WT and <i>p53^−/−</i> FDM cell lines (n = number of cell lines) were treated for 24 h with a MEK inhibitor (MEKi) in the presence or absence of IL-3. Viability was determined by flow cytometric analysis of PI exclusion. Results show the mean +/− SEM of 4 independent experiments.</p

Proportion of expressed probes for each array.

<p>Proportion of expressed probes for each array.</p

Differential gene expression in WT and <i>p53^−/−</i> FDM samples.

<p>(A) RNA from three independent WT and <i>p53^−/−</i> FDM cell lines were analyzed with the use of the 6-chip Illumina expression array. The heatmap depicts the expression of the top 30 differentially expressed genes according to the adjusted P value in WT and <i>p53^−/−</i> samples. (B) Differentially expressed genes highly expressed in <i>p53^−/−</i> compared to WT samples with a logFC change of greater than 2 are shown. (C) Differentially expressed genes highly expressed in WT compared to <i>p53^−/−</i> samples with a logFC change of greater than 2 are shown. Asterisks show the p53-dependent gene CDKN1A (p21).</p

Gene set enrichment analysis of WT and <i>p53^−/−</i> expression array data.

<p>(A) Gene Set Enrichment Analysis (GSEA) of the WT and <i>p53^−/−</i> gene lists at the various time points. GSEA was conducted using all genes that were considered expressed in the array, based on their detection p-value. A p-value of less than 0.05 was considered significant. (B) (C) Each diamond represents an individual probe for significantly differentially expressed kinases (B) or transcription factors (C) in WT and <i>p53^−/−</i> FDM cells cultured in IL-3.</p

Differential pathway expression in WT samples after IL-3 loss.

<p>(A) Three independent WT and <i>p53^−/−</i> FDM cell lines were culture with or without IL-3 for 6 h. RNA was extracted and expression array was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031428#pone-0031428-g001" target="_blank">Figure 1A</a>. The heat map shows significant changes in expression after IL-3 deprivation in WT cells and dots indicate the various SPIA pathways these represent. 31 differentially expressed genes were active in these pathways, with 24 being highly expressed at time zero and 12 highly expressed at 6 h IL-3 withdrawal. Significant pathways are shown (FDR<0.1). (B) Comparison of SPIA of array results from WT and <i>p53^−/−</i> samples after IL-3 withdrawal. The ID is the KEGG ID, pSize indicates the number of genes in the KEGG pathway, NDE is the number of differentially expressed genes found within the pathway and pGFdr is the False Discovery pathways (FDR<0.1). Significant pathways are shown.</p

Targeting acute myeloid leukemia by dual inhibition of PI3K signaling and Cdk9-mediated Mcl-1 transcription

Resistance to cell death is a hallmark of cancer and renders transformed cells resistant to multiple apoptotic triggers. The Bcl-2 family member, Mcl-1, is a key driver of cell survival in diverse cancers, including acute myeloid leukemia (AML). A screen for compounds that downregulate Mcl-1 identified the kinase inhibitor, PIK-75, which demonstrates marked proapoptotic activity against a panel of cytogenetically diverse primary human AML patient samples. We show that PIK-75 transiently blocks Cdk7/9, leading to transcriptional suppression of MCL-1, rapid loss of Mcl-1 protein, and alleviation of its inhibition of proapoptotic Bak. PIK-75 also targets the p110&alpha; isoform of PI3K, which leads to a loss of association between Bcl-xL and Bak. The simultaneous loss of Mcl-1 and Bcl-xL association with Bak leads to rapid apoptosis of AML cells. Concordantly, low Bak expression in AML confers resistance to PIK-75&ndash;mediated killing. On the other hand, the induction of apoptosis by PIK-75 did not require the expression of the BH3 proteins Bim, Bid, Bad, Noxa, or Puma. PIK-75 significantly reduced leukemia burden and increased the survival of mice engrafted with human AML without inducing overt toxicity. Future efforts to cotarget PI3K and Cdk9 with drugs such as PIK-75 in AML are warranted