Leukemia stem cells (LSCs) are believed to have more distinct vulnerabilities than the bulk acute myeloid leukemia (AML) cells, but their rarity and the lack of universal markers for their prospective isolation hamper their study. We report that genetically clonal induced pluripotent stem cells (iPSCs) derived from an AML patient and characterized by exceptionally high engraftment potential give rise, upon hematopoietic differentiation, to a phenotypic hierarchy. Through fate-tracking experiments, xenotransplantation, and single-cell transcriptomics, we identify a cell fraction (iLSC) that can be isolated prospectively by means of adherent in vitro growth that resides on the apex of this hierarchy and fulfills the hallmark features of LSCs. Through integrative genomic studies of the iLSC transcriptome and chromatin landscape, we derive an LSC gene signature that predicts patient survival and uncovers a dependency of LSCs, across AML genotypes, on the RUNX1 transcription factor. These findings can empower efforts to therapeutically target AML LSCs.
Epstein Barr virus (EBV) plays a causal role in some diseases, including infectious mononucleosis, lymphoproliferative diseases and nasopharyngeal carcinoma. Detection of EBV infection has been shown to be a useful tool for diagnosing EBV-related diseases. In the present study, we compared the performance of molecular tests, including fluorescence in situ hybridization (FISH) and EBV real-time PCR, to those of serological assays for the detection of EBV infection.Thirty-eight patients with infectious mononucleosis (IM) were enrolled, of whom 31 were diagnosed with a mild type, and seven were diagnosed with IM with haemophagocytic lymphohistiocytosis and chronic active EBV infection. Twenty healthy controls were involved in the study. The atypical lymphocytes in peripheral blood were detected under a microscope and the percentage of positive cells was calculated. EBV DNA load in peripheral blood was detected using real-time PCR. The FISH assay was developed to detect the EBV genome from peripheral blood mononuclear cells (PBMC). Other diagnosis methods including the heterophil agglutination (HA) test and EBV-VCA-IgM test, to detect EBV were also compared. SPSS17.0 was used for statistical analysis.In all, 5-41% atypical lymphocytes were found among the PBMC in mild IM patients, whereas 8-51% atypical lymphocytes were found in IM patients with haemophagocytic lymphohistiocytosis and chronic active EBV infection patients. There was no significant difference in the ratios of atypical lymphoma between patients of the different types. We observed that 71.2% of mild IM patients and 85.7% of IM patients with haemophagocytic lymphohistiocytosis and chronic active EBV infection patients were positive for EBV-VCA-IgM. EBV-VCA-IgM was negative in all healthy control subjects. In addition, 67.1% of mild IM patients tested heterophile antibody positive, whereas 71.4% of IM patients with haemophagocytic lymphohistiocytosis and chronic active EBV infection tested positive. EBV DNA detected using real-time PCR was observed in 89.5% of these IM patients. The EBV genome was detected by the FISH assay in 97.4% of the IM patients. The EB viral loads detected by FISH and real-time PCR increased with the severity of IM. The EBV genome was detected in almost all the PBMC of IM with haemophagocytic lymphohistiocytosis and chronic active EBV infection patients.Molecular tests, including FISH and EBV real-time PCR, are more sensitive than serological assays for the detection of EBV infection. The FISH assay detecting EBV copies in unfractionated whole blood is preferable and superior to plasma real-time PCR in its reflection of the absolute viral burden circulating in the patients.
Abstract AML1-ETO (AE) is a fusion transcription factor, generated by the t(8;21) translocation, that functions as a leukemia promoting oncogene. Here, we demonstrate that TATA-Box Binding Protein Associated Factor 1 (TAF1) associates with K43 acetylated AE and this association plays a pivotal role in the proliferation of AE-expressing acute myeloid leukemia (AML) cells. ChIP-sequencing indicates significant overlap of the TAF1 and AE binding sites. Knockdown of TAF1 alters the association of AE with chromatin, affecting of the expression of genes that are activated or repressed by AE. Furthermore, TAF1 is required for leukemic cell self-renewal and its reduction promotes the differentiation and apoptosis of AE+ AML cells, thereby impairing AE driven leukemogenesis. Together, our findings reveal a role of TAF1 in leukemogenesis and identify TAF1 as a potential therapeutic target for AE-expressing leukemia.
Abstract Hormone positive breast cancer remains one of the leading causes of cancer mortality in women. Despite the advancement in endocrine therapy and the addition of cyclin dependent kinase (CDK) 4/6 inhibitors, patients eventually become refractory to treatment creating a desperate need for the development of alternative therapies. Previously, we discovered HACE1, the E3 ubiquitin ligase that targets the small GTPase Rac1 for proteasomal mediated degradation. HACE1 is commonly lost in estrogen receptor (ER) positive and HER2 positive breast cancers and results in the hyperactivation of the Rac signaling pathway. Here, we show that Rac1 resides in the estrogen receptor transcriptional complex and acts as a critical estrogen receptor transcriptional co-activator. The dependency of Rac on ER transcription is further exemplified by the treating ER+ breast cancer cells with a Rac inhibitor where transcriptional profiling identified ER target genes were preferentially down-regulated. Thus, we confirm the dependency of Rac1 in ER+ breast cancers and show that Rac1 is necessary for estrogen receptor transcriptional activity. Importantly, we show that a novel Rac1 inhibitor is capable of reducing estrogen receptor transcriptional activity as well as impairing cellular proliferation and migration. Moreover, Rac inhibition is shown to enhance the anti-tumor effects of fulvestrant both in vitro and in vivo, including in a patient-derived-xenograft (PDX) model of ER+ disease. Lastly, we demonstrate that the administration of a novel Rac inhibitor is well-tolerated after chronic dosing in both small and large animal species. Together these data suggest a new potential pathway for treating hormone dependent breast cancer. Citation Format: Erik T Goka, Jun Sun, Dayrelis T Mesa Lopez, Marc E Lippman. Rac inhibition as an alternative approach to treating hormone dependent breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-13.
Abstract BACKGROUND: It is estimated that 67% of epithelial OVCAs are estrogen receptor (ER) positive. However, the response to anti-estrogen therapy in OVCA remains marginal. The Ras/Raf/MEK/MAPK pathway is hyperactivated in 40% of OVCAs. We have previously shown that estrogens further activate kinases such as Src, ER and Src kinase binding in the cytoplasm suggesting a non-genomic role or ER in OVCA. We postulated that estrogens further activate MAPK signaling and combination ER blockade with MEK inhibition would block cross-talk and increase the efficacy of ER blockade. METHODS: The effects of treatment with MEK inhibitor (AZD6244) and anti-estrogen (Fulvestrant), each alone or together, on cell cycle and cell survival were evaluated in ER+ OVCA lines in vitro. Drug effects on xenograft tumor growth were assayed in vivo in NOD/SCIDs. Reverse phase protein lysate array (RPPA) analysis and gene expression analysis (GEA) were performed to evaluate biomarkers of drug response. Finally, a previously reported MAPK gene signature identified in breast cancer was analyzed in the OVCA lines treated with AZD6244 and combination treatment. And using alteration of gene expression upon MEKi treatment as suggestive of MAPK regulation, we define a MAPK gene signature originating from ovarian cancer cells. RESULTS: RPPA analysis of high grade serous tumors from the TCGA (n=408) demonstrates that over 70% of tumors have phosphorylated MEK and MAPK, and patients with ER+ cancers and high pMAPK or pMEK (top50%), have a worse overall survival than those with low pMAPK or pMEK. Estrogen (E2) increases phosphorylation of MEK in ER+ OVCA cells. Fulvestrant caused minimal growth arrest after treatment demonstrating intrinsic resistance. AZD6244 caused loss of pMAPK, partial G1 cell cycle arrest and a modest increase in p27 levels in a dose dependent manner after treatment. However, responsiveness of OVCA cells to fulvestrant increased by addition of AZD6244 in vitro, with synergistic cell cycle arrest mediated by p27 binding to Cyclin E/cdk2 and much greater inhibition of MAPK activity. Gene enrichment analysis showed an increase in the ERB4/MAPK gene set with Fulv alone and the addition of AZD6244 showed that the top 20 gene sets downregulated were all related to replication and cell cycle (ie FOXM1, CyclinE). RPPA confirmed that combination was more effective in decreasing cell cycle promoting proteins (ie FOXM1, Cyclin B1) and upregulating p27. AZD6244 treatment of OVCA lines resulted in differential expression of about ¼ of the breast cancer defined MAPK gene expression signature, and of these, fulvestrant addition to MEK inhibition (MEKi) differentially affected 19 genes, reflective of these being E2 regulated genes. Similarly, of the total MEKi affected genes, a subset were differentially regulated by the addition of fulvestrant indicating putative E2 regulation underlying these genes. Xenograft data showed the greatest decrease in tumor volume with the drug combination compared to either drug alone. CONCLUSION: Given the majority of primary OVCAs express high MEK/MAPK activity may underlie failure of anti-estrogen therapy. MEK inhibition reverses anti-estrogen resistance in our OVCA models. These data support further pre-clinical and clinical evaluation of combined fulvestrant and MEK inhibition in OVCA. Citation Format: K. Hew, P. Miller, J. Sun, Z. Wei, G. Zhang, Y. Lu, G. Mills, J. Slingerland, MD, PhD, D. El-Ashry, F. Simpkins. MEK inhibition reverses antiestrogen resistance in ovarian cancer (OVCA) via alteration of cell cycle pathways and MAPK/estrogen regulated gene expression [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS31.
Curcumin, a selective phosphorylase kinase inhibitor, is a naturally occurring phytochemical present in turmeric. Curcumin has been confirmed to have anti-inflammatory properties in addition to the ability to decrease the expression of pro-inflammatory cytokines in keratinocytes. The interleukin-23 (IL-23)/IL-17A cytokine axis plays a critical role in the pathogenesis of psoriasis. Here, we report that topical use of a curcumin gel formulation strongly inhibited imiquimod (IMQ)-induced psoriasis-like inflammation, the development of which was based on the IL-23/IL-17A axis. IMQ-induced epidermal hyperplasia and inflammation in BALB/c mouse ear was significantly inhibited following curcumin treatment. Real-time PCR showed that mRNA levels of IL-17A, IL-17F, IL-22, IL-1β, IL-6 and TNF-α cytokines were decreased significantly by curcumin in ear skin, an effect similar to that of clobetasol. In addition, we found that curcumin may enhance the proliferation of epidermis γδ T cells but inhibit dermal γδ T cell proliferation. We inferred that curcumin was capable of impacting the IL-23/IL-17A axis by inhibiting IL-1β/IL-6 and then indirectly down-regulating IL-17A/IL-22 production. In conclusion, curcumin can relieve the IMQ-induced psoriasis-like inflammation in a mouse model, similar to the effects of clobetasol. Therefore, we have every reason to expect that curcumin will be used in the treatment of psoriasis in the future.
The estrogen receptor (ER) binds to estrogen-responsive elements (EREs) to activate gene transcription. The best characterized EREs are located in proximal gene promoters, but recent data indicate that only a minority of ER binding sites lie within proximal promoter regions. GREB1 (gene regulated by estrogen in breast cancer 1) is an ER target gene that regulates estrogen-induced proliferation in breast cancer cells. We identified three consensus EREs, located at -21.2, -9.5, and -1.6 kb upstream of the closest GREB1a transcription start site that appear to mediate long-range GREB1 gene activation by ER. All three ERE sites nucleate ER, steroid receptor coactivator-3 (SRC-3), and RNA polymerase II (Pol II) and undergo histone acetylation in response to estradiol. Estrogen-stimulated ER binding at all three EREs was cyclic and synchronous. SRC-3 and Pol II recruitment to all three EREs was activated by estrogen but not tamoxifen. In contrast, estrogen stimulated only Pol II and not ER or SRC-3 recruitment to the GREB1 core promoter regions. Long-range histone acetylation, centered on the three ERE motifs and the GREB1 core promoters, was observed in response to estrogen but not to tamoxifen. These data suggest that estrogen-stimulated GREB1 transcription may involve coordinated ER binding to all three distal consensus ERE motifs. Long-range activation by ER acting at multiple EREs may be more common than previously appreciated.
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