Tfeb Links MYC Signaling to Epigenetic Control of Acute Myeloid Leukemia Cell Death and Differentiation

MYC gene amplification and somatic mutations are frequent in both adult and pediatric AML although how MYC drives and contributes to the development and maintenance of AML has not been resolved. Transcription factor EB (TFEB) is a master regulator of genes that control autophagy and lysosome biogenesis, a central catabolic recycling pathway that regulates cell survival. Given the oncogenic effects of MYC in AML and that the induction of autophagy compromises AML cell growth and survival, we tested if the oncogenic effect of MYC depends on its suppression of TFEB transcription programs in AML. In support of this hypothesis, inducible MYC expression in K562 and THP-1 leukemia cells was sufficient to suppress expression of TFEB and its target genes. Further and conversely, MYC knockdown in NB4 AML cells provoked increased expression of TFEB mRNA and protein, as well as increased expression of TFEB target genes. Notably, dose response studies demonstrated that expression of TFEBS211A, constitutively nuclear form of TFEB that is refractory to control by mTORC1 signaling, dramatically impairs proliferation of HL60, OCI-AML2 and OCI-AML3 AML cells. In addition, induction of TFEBS211A provoked rampant apoptosis. Of important, overexpression of TFEBS211A in HL-60 and OCI-AML3 cells was also sufficient to promote monocytic and granulocytic differentiation, as judged by morphological changes and the acquisition of mature monocytic and granulocytic markers including CD11b, Gr1, and CD15. To identify TFEB targets that might contribute to myeloid/granulocytic differentiation, we performed RNA-seq analysis of HL60 leukemia cells engineered to inducibly express the TFEBS211A transgene. Using a cut-off of fold change>4 with q<0.01, a total of 1152 genes were differentially regulated following the induction of TFEBS211A in HL60 cells. As expected, this included the robust induction of nearly all TFEB target genes associated with the autophagy-lysosome pathway, but also of STAT1, KLF4, KLF6, CEBPB, CSF1 and GATA2 genes that are necessary and/or sufficient to provoke terminal monocytic and granulocytic differentiation of AML cells. Surprisingly, among genes induced by TFEB in HL60 cells is IDH1, which catalyzes the production of α-ketoglutarate (α-KG), a required substrate of the TET family of dioxygenases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). In particular, TFEBS211A expression provoked increases in levels of α-KG and significant increases in global levels of 5hmC in genomic DNA of HL60 leukemia cells both ex vivo and in vivo. Furthermore, TFEB-mediated induction of IDH1/2 mRNA and protein, and of IDH1 promoter activity, was antagonized by inducible expression of MYC. To assess the global effects of TFEB on 5mC/5hmC landscapes, we performed paired reduced representation bisulfite (BS)- and oxidation bisulfite (oxBS)-sequencing. As predicted, TFEBS211A induced both loss and gains of 5mC, but there were more losses (n = 722) than gains (n = 459) across all 22 chromosomes. Quite remarkably, and consistent with TFEB-provoked increases of 5hmC signals, TFEBS211A exclusively induced 5hmC gains (in a total of 863 genes), and 37% and 36% of these 5hmC gains occurred in promoter regions and CpG islands, and across all 22 chromosomes. Comparison of BS- and oxBS-seq versus RNA-seq analyses of HL60 cells expressing TFEB revealed significant changes in mRNA levels and concomitant differential changes in 5mC and 5hmC marks in KLF4, KLF6, STAT3, TP73, andFOXO1 that have pivotal roles in controlling myeloid cell differentiation and death. Collectively, these findings demonstrate that MYC suppresses TFEB expression and function in AML cells, and that TFEB functions as a tumor suppressor that provokes AML cell differentiation and death. Strikingly, these responses rely on epigenetic control, where TFEB directly induces the transcription of IDH1 and IDH2 to provoke global hydroxylation of 5-methylcytosine and the expression of genes that drive terminal differentiation and apoptosis. Thus, a MYC-TFEB-IDH1/2-TET2 circuit controls AML cell fate. Disclosures Murphy: Merck: Research Funding; Puma Biotech: Research Funding. Ballabio:CASMA Therapeutics: Other: Co-Founder. Kaufmann:Takeda Pharmaceuticals: Research Funding.

Download Full-text

Regulation of Ketogenic Enzyme HMGCS2 by Wnt/β-catenin/PPARγ Pathway in Intestinal Cells

Cells ◽

10.3390/cells8091106 ◽

2019 ◽

Vol 8 (9) ◽

pp. 1106 ◽

Cited By ~ 3

Author(s):

Ji Tae Kim ◽

Chang Li ◽

Heidi L. Weiss ◽

Yuning Zhou ◽

Chunming Liu ◽

...

Keyword(s):

Cell Differentiation ◽

Cell Lines ◽

Cancer Cell ◽

Target Genes ◽

Cancer Cell Lines ◽

Intestinal Cell ◽

Mrna Levels ◽

Intestinal Epithelial ◽

Human Colon Cancer ◽

Intestinal Organoids

The Wnt/β-catenin pathway plays a crucial role in development and renewal of the intestinal epithelium. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme in the synthesis of ketone body β-hydroxybutyrate (βHB), contributes to the regulation of intestinal cell differentiation. Here, we have shown that HMGCS2 is a novel target of Wnt/β-catenin/PPARγ signaling in intestinal epithelial cancer cell lines and normal intestinal organoids. Inhibition of the Wnt/β-catenin pathway resulted in increased protein and mRNA expression of HMGCS2 and βHB production in human colon cancer cell lines LS174T and Caco2. In addition, Wnt inhibition increased expression of PPARγ and its target genes, FABP2 and PLIN2, in these cells. Conversely, activation of Wnt/β-catenin signaling decreased protein and mRNA levels of HMGCS2, βHB production, and expression of PPARγ and its target genes in LS174T and Caco2 cells and mouse intestinal organoids. Moreover, inhibition of PPARγ reduced HMGCS2 expression and βHB production, while activation of PPARγ increased HMGCS2 expression and βHB synthesis. Furthermore, PPARγ bound the promoter of HMGCS2 and this binding was enhanced by β-catenin knockdown. Finally, we showed that HMGCS2 inhibited, while Wnt/β-catenin stimulated, glycolysis, which contributed to regulation of intestinal cell differentiation. Our results identified HMGCS2 as a downstream target of Wnt/β-catenin/PPARγ signaling in intestinal epithelial cells. Moreover, our findings suggest that Wnt/β-catenin/PPARγ signaling regulates intestinal cell differentiation, at least in part, through regulation of ketogenesis.

Download Full-text

Rig-I Activates Expression of Interferon-Stimulated Genes (ISGs) and Inhibits the Proliferation of Acute Myeloid Leukemia Cells

Blood ◽

10.1182/blood.v112.11.2846.2846 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2846-2846 ◽

Cited By ~ 1

Author(s):

Nan-Nan Zhang ◽

Lei Chen ◽

Wu Zhang ◽

Xian-Yang Li ◽

Lin-Jia Jiang ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Leukemia Cells ◽

Mrna Levels ◽

Terminal Part ◽

Granulocytic Differentiation ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

Abstract Acute promyelocytic leukemia (APL) is initiated by the formation of PML/RARα oncogenic fusion protein, a potent transcriptional repressor. Retinoid acid (RA) at pharmacological dosage can physically bind to the PML/RARα protein, ushering in the unfolding of downstream programs normally regulated by the wild type RARα. However, through what particular regulatory pathways RA inhibits APL malignant hematopoiesis has remained largely obscured. Rig-I is one of the genes whose mRNA levels were highly up-regulated, along with all-trans-RA (ATRA)-induced terminal granulocytic differentiation of APL cell line NB4 cells in vitro. Based on the analysis of a Rig-I−/− mouse model, recently we have reported a critical regulatory role of Rig-I in normal granulopoiesis. To understand the functional contribution of Rig-I induction in RA-mediated leukemia cell differentiation, we converted a pair of previously reported Rig-I RNAi-duplex sequences into a miR30a-based small hairpin-encoding sequence, which was expressed under the CMV enhancer/promoter within a lentiviral vector. As expected, Rig-I shRNAmir30 infection induced a significant knockdown of Rig-I protein level, and accordingly its delivery into HL-60 cells partially inhibited ATRA-induced granulocytic differentiation, growth inhibition/cell cycle arrest and apoptosis induction, suggesting that Rig-I upregulation participates in RA-induced granulocytic differentiation of acute myeloid leukemia cells. In order to investigate the effect of Rig-I induction on the proliferation of APL cells in vivo, we transduced PML/RARα-harboring leukemic cells with vector or Rig-I-expressing retrovirus, and then transplanted these cells into the syngeneic mice. The vector-transduced APL cells readily expanded in vivo, but the proliferation of Rig-I-transduced cells was apparently prohibited. Moreover, we found that the forced expression of Rig-I induced the expression of numerous ISGs in APL cells, which was recapitulated by the transduction of the C terminal part of Rig-I, but not by the N terminal part. In line with this, during the in vitro short-term culture post-IFNγ or IFNα stimulation, Stat1 phosphorylation at p701 in Rig-I−/− granulocytes was significantly inhibited. In parallel, the induction of multiple ISGs by IFNs was also significantly impaired. In conclusion, our findings indicate that the Rig-I induction inhibited APL reconstitution potentially through up-regulating a number of ISGs via regulating Stat1Tyr701 phosphorylation.

Download Full-text

BACH2 Is Required for Pre-B Cell Receptor Checkpoint Control and p53-Dependent Tumor Surveillance

Blood ◽

10.1182/blood.v120.21.1300.1300 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1300-1300

Author(s):

Srividya Swaminathan ◽

Huining Kang ◽

Richard C. Harvey ◽

Chuanxin Huang ◽

Maike Buchner ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

Clinical Outcome ◽

B Cell ◽

Leukemia Cells ◽

Mrna Levels ◽

B Cell Differentiation ◽

Childhood All ◽

Cell Clones ◽

Favorable Clinical Outcome

Abstract Abstract 1300 Background: BACH2 (BTB and CNC homology 1, basic leucine zipper transcription factor 2) is required for class-switch recombination and somatic hypermutation of immunoglobulin genes during affinity maturation of mature germinal center B cells. We and others found that BACH2 is strongly upregulated in BCR-ABL1-transformed acute lymphoblastic leukemia (Ph+ ALL) cells upon treatment with tyrosine kinase inhibitors (TKI). Results: Bach2 mRNA levels are significantly lower in Ph+ ALL (n=72) compared to normal human bone marrow pre-B cells (n=10). Studying gene expression in a clinical trial for children with high risk ALL (Children's Oncology Group, P9906; n=207), we found in a multivariate analysis that high Bach2 levels at the time of diagnosis represents an independent predictor of favorable clinical outcome (negative MRD at day and higher overall and relapse-free survival; p<0.0001). We next studied 49 sample pairs from patients with childhood ALL at diagnosis and relapse. In 44 of these sample pairs, the relapse sample showed drastically reduced mRNA levels of Bach2 (p=0.019), suggesting that loss of BACH2 expression is associated with relapse of childhood ALL. A comparison of the methylation status of BACH2 promoter of normal pre-B cells (n=5), with Ph+ ALL cells (n=70) revealed that CpG islands in the BACH2 promoter were heavily hypermethylated in the leukemia samples. These findings are also consistent with genomic analyses on patient derived samples and the identification of small deletions at 6q15 in 4 of 11 cases of childhood ALL cases that all span the BACH2 locus. To study the role of Bach2 in pre-B ALL in a genetic experiment, we transformed pre-B cells from Bach2−/− mice with BCR-ABL1. We observed that Bach2−/− normal pre-B cells lack the ability to counterselect pre-B cell clones that failed to undergo successful V(D)J rearrangement. In the absence of Bach2, a significant number of B cells survive even though they failed to rearrange immunoglobulin heavy chain genes. Besides this unexpected role in early B cell differentiation, quantitative RT-PCR and Western blot confirmed that BACH2 is also required for expression of the tumor suppressors Cdkn2a (Arf), p53 and Btg2. Consistent with extremely low protein levels of Arf and p53 in Bach2−/− leukemia cells, Bach2−/− ALL cells are more resistant to TKI-treatment, more actively proliferating (increased S-phase; p=0.02) and exhibit a ∼90-fold increased ability to form colonies in methyl cellulose (p=0.001). Studying Cre-mediated inducible deletion of p53 in p53-fl/fl leukemia cells, we found that Bach2-induced tumor suppression is largely dependent on p53 function. Forced overexpression of Myc results in oncogene-induced senescence (OIS) followed by apoptosis. Whereas Bach2+/+ leukemia cells are non-permissive to forced Myc expression and die within four days after Myc induction, Bach2−/− ALL cells tolerate forced expression of Myc and evade OIS and subsequent cell death. Similarly, overexpression of Myc alone fails to transform Bach2+/+ pre-B cells. By contrast, retroviral overexpression of Myc results in rapid transformation and growth factor-independence of Bach2−/− pre-B cells. Bach2−/− Myc-high pre-B cells cause fatal leukemia in 100% of recipient mice within 22 days, whereas all mice that received Bach2+/+ Myc-high pre-B cells survived without signs of disease until day 67, when all mice were sacrificed and analyzed for MRD by flow cytometry and PCR. No evidence of MRD was detected in most mice injected with Bach2+/+ Myc-high pre-B cells. Three mice had positive MRD PCR findings, however, at 4 log orders below findings in mice injected with Bach2−/−Myc-high pre-B cells. Conclusions: Our findings identify Bach2 as a novel tumor suppressor upstream of p53 in pre-B ALL. Bach2 is a regulator of negative selection during normal pre-B cell differentiation but also limits excessive proliferation of pre-B cell clones by induction of oncogene-induced senescence and activation of p53. In addition, our multivariate analyses identify high expression levels of Bach2 as powerful predictor of favorable clinical outcome in children, which may be useful in future approaches for risk stratification. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Transcription Factor Bach1 and Bach2 Control Common Myeloid Progenitor Cell Differentiation Under Infectious Stimuli

Blood ◽

10.1182/blood.v126.23.1164.1164 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1164-1164

Author(s):

Hiroki Kato ◽

Ari Itoh-Nakadai ◽

Risa Ebina-Shibuya ◽

Masahiro Kobayashi ◽

Mitsuyo Matsumoto ◽

...

Keyword(s):

Gene Expression ◽

Cell Differentiation ◽

Progenitor Cells ◽

Research Funding ◽

Myeloid Cell ◽

Gene Set Enrichment Analysis ◽

Mrna Levels ◽

Myeloid Progenitor ◽

Dd Mice ◽

Stimulation Of

Abstract Background: Erythrocyte and granulocyte/macrophage develop from common myeloid progenitor (CMP) (Akashi et al., 2000). Differentiation of hematopoietic progenitor cells is precisely controlled by multiple transcription factors, among which GATA1, C/EBPα, C/EBPβ and Spi-C play pivotal roles in erythrocyte and granulocyte/macrophage differentiation (Mancini et al., 2012; Pongubala et al., 2008; Hirai et al., 2006; Haldar et al., 2014). However, the mechanism by which the differentiation of CMP controlled under infectious condition has been unclear. Bach1 and Bach2 belong to the basic region-leucine zipper family and recognize Maf-recognition elements (Oyake et al., 1996). They promote B cell development by repressing the myeloid genes such as Cebpb and Spic in common lymphoid progenitor cells (Itoh-Nakadai et al., 2014). In addition, Bach1 regulates several target genes related to iron/heme homeostasis such as globin genes and hemeoxygenase-1, and Bach2 may similarly regulate these genes (Igarashi, 2014). Therefore, it is expected that both Bach1 and Bach2 play redundant roles in erythropoiesis. To figure out their roles in erythroid and myeloid cell differentiation, we performed hematological and transcriptomics analyses using Bach1-/- Bach2-/- (double-deficient; DD) mice. Methods: The generation of DD mice on the C57BL/6J background and Bach2 reporter mice with red fluorescent protein coding cDNA inserted in the Bach2 locus were described previously (Itoh-Nakadai et al., 2014). Mice between 8-12 weeks old were analyzed in the present study. Bone marrow (BM) cells were stained with specific combinations of antibodies to identify erythroid/myeloid progenitor and mature cells (Sheila et al., 2008; Cornelis et al., 2007; Socolovsky et al., 2001). Flow cytometry analysis and cell sorting were performed by using FACSAriaⅡ(BD) and FlowJo software (TreeStar). For infectious simulation of CMP, sorted CMPs were incubated with 1μg/ml LPS (Sigma) for 48h and RNA was purified with RNeasy micro kit (Qiagen). Quantitative PCR by using SuperscriptⅢ reverse transcriptase (Invitrogen) and Light Cycler system (Roche) was performed according to manufacturer's instructions. Microarray analysis by using Sure-Print G3 mouse GE microarray slide (Agilent) was performed as previously described (Itoh-Nakadai et al., 2014) and the results were analyzed by using GeneSpring software (Agilent). We used Gene Set Enrichment Analysis (GSEA) to interpret gene expression data (Subramanian et al., 2005; Mootha et al., 2003). LPS stimulation (50 μg/body) of mice was performed as previously described (Ryan et al., 2008). Data were analyzed by the two-sided Student's t-test and p - values of <0.05 were considered statistically significant. Results: DD mice show mild normocytic anemia compered to wild-type (WT), Bach1-/-, and Bach2-/- mice (hemoglobin; 14.4±0.2, 14.0±0.3, 13.5±0.3 and 11.9±0.7 g/dl, for WT, Bach1-/-, Bach2-/- and DD, respectively, p<0.05 for comparison between DD and other genotypes, n=7). Immature and mature erythroblast populations were significantly decreased in BM of DD (immature; 25.8±1.78, 15.6±1.4, mature; 27.6±3.3, 17.4±2.3×106/body for WT and DD, respectively, p<0.05, n=6). Megakaryocyte-erythroid progenitor (MEP)/granulocyte-monocyte progenitor (GMP) ratio was significantly decreased in BM of DD (MEP/GMP: 0.13±0.01, 0.07±0.01 for WT and DD, respectively, p<0.05, n=5). Bach2 expression was detected in CMP, MEP and even GMP by using Bach2-RFP mice. LPS stimulation of WT CMP significantly decreased mRNA levels of Bach1, Bach2 and Gata1. On the other hand, Cebpb and Spic mRNA levels were significantly increased. LPS stimulation of WT mice induced significant increase of granulocyte and decrease of erythrocyte and B lymphocyte in BM, which was consistent with previous reports. It was also shown that LPS stimulation significantly decreased MEP/ GMP ratio. According to the clustering analysis of the microarray data of CMP sorted from WT and DD mice, they showed clearly different expression profiles. GSEA showed that CMP of DD skewed to myeloid cell lineage and lost the erythroid gene expression compared to WT. Conclusions: Bach1 and Bach2 control the differentiation of CMP to erythroid cell or myeloid cell by repressing myeloid genes such as Cebpb and Spic. Infectious stimuli may promote myeloid cell differentiation by reducing the expression of Bach1 and Bach2 in CMP. Disclosures Fujiwara: Chugai Pharmaceutical CO., LTD: Research Funding. Harigae:Chugai Pharmaceutical CO., LTD: Research Funding.

Download Full-text

FLT3 Inhibition Downregulates EZH2 in AML and Promotes Myeloid Differentiation

Blood ◽

10.1182/blood-2021-147313 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 785-785

Author(s):

Pamela J. Sung ◽

Simone Sidoli ◽

Simone S. Riedel ◽

Katarzyna Kulej ◽

Hongbo Xie ◽

...

Keyword(s):

Research Funding ◽

Progenitor Cell ◽

Target Genes ◽

Expression Profiles ◽

Colony Morphology ◽

Leukemic Cells ◽

Myeloid Differentiation ◽

Granulocytic Differentiation ◽

Induced Differentiation ◽

Cell State

Abstract Internal tandem duplication mutations in the Fms-like tyrosine kinase 3 (FLT3-ITD) are frequently recurring in AML and confer a poor prognosis. FLT3 inhibitors (FLT3i) such as gilteritinib are efficacious in relapsed AML. Clinical responses to FLT3i include myeloid differentiation of the FLT3-ITD clone in about 50% of patients. How FLT3i induce this response in a subset of patients is unknown. The FLT3i-induced differentiation response seen in clinical trials has not previously been demonstrated in animal models. We modeled FLT3i-induced differentiation in murine Flt3 ITD/ITDDnmt3a -/- AML model (Meyer et al., Cancer Discovery, 2016). Treatment with FLT3i in vitro accelerated differentiation of cKIT+ leukemic splenocytes as assessed by colony morphology in serial re-plating assays. To characterize the differentiation response in vivo, we transplanted CD45.2+ leukemic splenocytes from moribund mice into sub-lethally irradiated healthy congenic CD45.1+ mice. After confirmation of engraftment at 2 weeks post-irradiation, mice were treated with vehicle or gilteritinib for 4 weeks. Animals treated with gilteritinib demonstrated increased neutrophil and decreased stem/progenitor cell populations, recapitulating the clinically observed increase in granulocytic differentiation of the FLT3-ITD clone. We next sought to understand the molecular mechanism of FLT3i-induced differentiation. We used a proteomic-based screen in a human AML cell line treated with FLT3i to identify novel targets of FLT3-ITD that could be potential mediators of the differentiation response. We identified downregulation of Enhancer of Zeste Homolog 2 (EZH2), the catalytic component of the Polycomb Repressive Complex 2 (PRC2). EZH2 and PRC2 were previously shown to be required for leukemic maintenance in mouse models of MLL-AF9 AML. We treated murine Flt3 ITD/ITDDnmt3a -/- cKIT+ leukemic splenocytes with FLT3i or the EZH1/2 inhibitor (EZH1/2i). Both promoted myeloid differentiation to similar degrees as assessed by colony morphology in this model. We hypothesized that FLT3-ITD regulates EZH2 to maintain leukemia cells in a stem/progenitor cell state. We, therefore, characterized the effect of FLT3i on PRC2 in more detail. We confirmed that FLT3i decreases EZH2 protein levels in FLT3-ITD cell lines and primary human AML within 24 hours of treatment as suggested by our proteomic data (Figure 1A-B). We found that the mechanism of EZH2 downregulation is complex with both transcriptional effects and a decrease in EZH2 protein half-life. ChIP-Seq for H3K27me3 demonstrated decreased peaks at the transcription start sites of PRC2 target genes (Figure 1C). RNA-Seq gene expression profiles of FLT3i- and EZH1/2i-treated human AML cells overlapped at 253 differentially expressed genes (Figure 1D). Critically, both FLT3i and EZH1/2i expression profiles enriched in differentiated myeloid cell gene signatures. Overall, we found that EZH2 is a novel, unexpected, and clinically relevant target of FLT3-ITD. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the FLT3i-induced differentiation response seen in patients. These data demonstrate that FLT3-ITD has at least two functions in leukemogenesis, the well described activation of signaling pathways, and second, a previously undefined, regulation of PRC2 to maintain a myeloid stem cell state. Our results may lead to improved approaches to therapy for FLT3 mutated AML. Figure 1 Figure 1. Disclosures Bernt: Syndax: Research Funding; Merck: Other: Spouse is an employee of Merck.. Carroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy.

Download Full-text

PKCδ Regulates Eukaryotic Initiation Factor eIF2α through PKR during Retinoic Acid-Induced Myeloid Cell Differentiation.

Blood ◽

10.1182/blood.v108.11.1928.1928 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1928-1928

Author(s):

Bulent Ozpolat ◽

Ugur Akar ◽

Magaly Barria ◽

Gabriel Lopez-Berestein

Keyword(s):

Retinoic Acid ◽

Cell Differentiation ◽

Protein Kinase ◽

Translation Initiation ◽

Myeloid Leukemia ◽

Mrna Translation ◽

Leukemia Cells ◽

Granulocytic Differentiation ◽

Nb4 Cells

Abstract Dysregulation of mRNA translation can contribute to malignant transformation. Translation initiation is a rate limiting step of mRNA translation and protein synthesis and plays a critical role in regulation of cell growth, proliferation and differentiation. We previously reported that ATRA induces translational suppression through multiple posttranscriptional mechanisms during terminal cell differentiation detected by proteomic analysis (Harris et al, Blood, 104 (5) 2004). Here we investigated the regulation of translation initiation and the role of eIF2α during terminal differentiation of myeloid leukemia cells. We found that ATRA and other granulocytic differentiation inducing agents, such as dimethyl sulfoxide (DMSO), arsenic trioxide (ATO) induce phosphorylation of eIF2α on serine 51 in promyelocytic leukemia (NB4) cells, indicating the suppression of translation initiation. However, monocytic/macrophagic differentiation of NB4 cells by phorbol 12-myristate 13-acetate (phorbol ester, PMA), or by ATRA in U937 and THP-1 myelomonoblastic myeloid leukemia (AML) cells, was not accompanied with induction of eIF2α phosphorylation. ATRA, ATO or DMSO-induced granulocytic differentiation closely correlated with induction of expression and phosphorylation/activation of protein kinase C-delta (PKCδ) on threonin 505 and serine 643 in NB4 cells. The specific PKCδ inhibitor, rottlerin, markedly inhibited ATRA-induced expression and phosphorylation (serin 51) of eIF2a in NB4 cells. Rottlerin reduced phosphorylation of eIF2α expression not only in the leukemia cells but also in solid tumor cells such as breast (MCF7) and pancreatic (Panc28) cancer cells. Because protein kinase R (PKR) has been shown to inhibit mRNA translation by inducing phosphorylation of eIF2α, we also examined whether this pathway is involved in ATRA-induced phosphorylation of eIF2α and whether it is downstream of PKCδ. We observed that ATRA induces expression and phosphorylation/activation of PKR in NB4 cells. Rottlerin inhibited ATRA-induced expression and activity of PKR , suggesting that activity of PKR is regulated by PKCδ in response to ATRA in NB4 cells. Overall, our data suggest that retinoic acid suppresses translation initiation through PKCδ/PKR/eIF2α pathway during granulocytic but not monocytic differentiation of acute myeloid leukemia cells. These results revealed a novel role of ATRA in granulocytic cell differentiation of myeloid cells. Because malignant cells usually have hyperactivated mRNA translation, targeting translational factors/regulators of initiation may offer new strategies for the treatment of myeloid leukemia cells.

Download Full-text

PKCδ Regulates Retinoic Acid-Induced Myeloid Cell Differentiation through Eukaryotic Initiation Factor Alpha (eIF2α).

Blood ◽

10.1182/blood.v110.11.1814.1814 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1814-1814

Author(s):

Bulent Ozpolat ◽

Ugur Akar ◽

Magaly Barria ◽

Gabriel Lopez-Berestein

Keyword(s):

Retinoic Acid ◽

Cell Differentiation ◽

Myeloid Leukemia ◽

Initiation Factor ◽

Leukemia Cells ◽

Eukaryotic Initiation Factor ◽

Granulocytic Differentiation ◽

Nb4 Cells ◽

Factor Alpha ◽

Expression And Activity

Abstract Overactivity of eukaryotic initiation factor-alpha (eIF2α) has been shown to be oncogenic and induces malignant transformation. Here we investigated the regulation and the role of eIF2α in the terminal differentiation of myeloid leukemia cells. We found that all-trans-retinoic acid (ATRA) and other granulocytic differentiation inducers, such as dimethylsulfoxide and arsenic trioxide inhibited activity of eIF2α by inducing serine 51 phosphorylation in promyelocytic leukemia cells (NB4). In contrast, activity of eIF2α was unaffected during ATRA-induced monocytic differentiation of U937 and THP-1 myelomonocytic cells and phorbol 12-myristate 13-acetate-induced monocytic/macrophagic differentiation of NB4 cells. Knockdown of eIF2α by RNA interference (siRNA) significantly inhibited (p<0.05) ATRA-induced differentiation, indicating that eIF2α is critical for the induction of granulocytic differentiation. ATRA-induced eIF2α phosphorylation was correlated with the expression and activity/phosphorylation (Thr505 and Ser643) of protein kinase Cδ (PKCδ) and eIF2a kinase PKR (Thr446). The specific PKCδ inhibitor Rottlerin significantly reduced phosphorylation of eIF2α, activity of PKR and blocked ATRA-induced granulocytic differentiation of NB4 cells (p<0.05). Knockdown of PKCδ by siRNA decreased PKR activity and increased eIF2α activity while knockdown of PKR increased eIF2α activity. We also observed that PKCδ regulates eIF2a activity in normal CD34+ bone marrow progenitor cells and breast and pancreatic cancer cell lines. Furthermore, we found that eIF2α regulated the expression and activity of important targets of ATRA, including c-myc, p21Waf1/Cip1, DAP5, GADD153, ATF-2, TG2, and p-P70S6K. In conclusion, our findings indicate that ATRA-induced granulocytic differentiation of myeloid leukemia cells is regulated by PKCδ through the activity of eIF2α, revealing a novel mechanism of granulocytic cell differentiation. Figure Figure

Download Full-text

Foxa1 and Foxa2 Regulate α-Cell Differentiation, Glucagon Biosynthesis, and Secretion

Endocrinology ◽

10.1210/en.2013-1843 ◽

2014 ◽

Vol 155 (10) ◽

pp. 3781-3792 ◽

Cited By ~ 22

Author(s):

Mounia Heddad Masson ◽

Caroline Poisson ◽

Audrey Guérardel ◽

Aline Mamin ◽

Jacques Philippe ◽

...

Keyword(s):

Gene Expression ◽

Cell Differentiation ◽

Target Genes ◽

Adult Mouse ◽

Glucagon Secretion ◽

Mrna Levels ◽

Forkhead Box ◽

Interfering Rna

Abstract The Forkhead box A transcription factors are major regulators of glucose homeostasis. They show both distinct and redundant roles during pancreas development and in adult mouse β-cells. In vivo ablation studies have revealed critical implications of Foxa1 on glucagon biosynthesis and requirement of Foxa2 in α-cell terminal differentiation. In order to examine the respective role of these factors in mature α-cells, we used small interfering RNA (siRNA) directed against Foxa1 and Foxa2 in rat primary pancreatic α-cells and rodent α-cell lines leading to marked decreases in Foxa1 and Foxa2 mRNA levels and proteins. Both Foxa1 and Foxa2 control glucagon gene expression specifically through the G2 element. Although we found that Foxa2 controls the expression of the glucagon, MafB, Pou3f4, Pcsk2, Nkx2.2, Kir6.2, and Sur1 genes, Foxa1 only regulates glucagon gene expression. Interestingly, the Isl1 and Gipr genes were not controlled by either Foxa1 or Foxa2 alone but by their combination. Foxa1 and Foxa2 directly activate and bind the promoter region the Nkx2.2, Kir6.2 and Sur1, Gipr, Isl1, and Pou3f4 genes. We also demonstrated that glucagon secretion is affected by the combined effects of Foxa1 and Foxa2 but not by either one alone. Our results indicate that Foxa1 and Foxa2 control glucagon biosynthesis and secretion as well as α-cell differentiation with both common and unique target genes.

Download Full-text

Synthetic De Novo Modeling of Human T-Cell Acute Lymphoblastic Leukemia Reveals HOXB Genes Drive Expansion of Leukemia Cells-of-Origin and Established Tumor Clones

Blood ◽

10.1182/blood-2018-99-120120 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1322-1322

Author(s):

Manabu Kusakabe ◽

Ann Chong Sun ◽

Kateryna Tyshchenko ◽

Rachel Wong ◽

Aastha Nanda ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Research Funding ◽

De Novo ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Rna Seq ◽

Cell Acute Lymphoblastic Leukemia

Abstract Mechanistic studies in human cancer have relied heavily on established cell lines and genetically engineered mouse models, but these are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts (PDX); however, as an experimental model these are hampered by their variable genetic background, logistic challenges in establishing and distributing diverse collections, and the fact they cannot be independently reproduced. We report here a completely synthetic, efficient, and highly reproducible means for generating T-cell acute lymphoblastic leukemia (T-ALL) de novo by lentiviral transduction of normal CD34+ human cord blood (CB) derived hematopoietic progenitors with a combination of known T-ALL oncogenes. Transduced CB cells exhibit differentiation arrest and multi-log expansion when cultured in vitro on OP9-DL1 feeders, and generate serially transplantable, aggressive leukemia when injected into immunodeficient NSG mice with latencies as short as 80 days (median 161 days, range 79-321 days). RNA-seq analysis of synthetic CB leukemias confirmed their reproducibility and similarity to PDX tumors, while whole exome sequencing revealed ongoing clonal evolution in vivo with acquisition of secondary mutations that are seen recurrently in natural human disease. The in vitro component of this synthetic system affords direct access to "pre-leukemia" cells undergoing the very first molecular changes as they are redirected from normal to malignant developmental trajectories. Accordingly, we performed RNA-seq and modified histone ChIP-seq on nascently transduced CB cells harvested from the first 2-3 weeks in culture. We identified coordinate upregulation of multiple anterior HOXB genes (HOXB2-B5) with contiguous H3K27 demethylation/acetylation as a striking feature in these early pre-leukemia cells. Interestingly, we also found coordinate upregulation of these same HOXB genes in a cohort of 264 patient T-ALLs (COG TARGET study) and that they defined a subset of patients with significantly poorer event-free survival (Log-rank p-value = 0.0132). Patients in the "HOXB high" subgroup are distinct from those with ETP-ALL, but are enriched within TAL1, NKX2-1, and "unknown" transcription factor genetic subgroups. We further show by shRNA-mediated knockdown that HOXB gene expression confers growth advantage in nascently transduced CB cells, established synthetic CB leukemias, and a subset of established human T-ALL cell lines. Of note, while there is prior literature on the role of HOXA genes in AML and T-ALL, and of HOXB genes in normal HSC expansion, this is the first report to our knowledge of a role for HOXB genes in human T-ALL despite over 2 decades of studies relying mostly on mouse leukemia and cell line models. The synthetic approach we have taken here allows investigation of both early and late events in human leukemogenesis and delivers an efficient and reproducible experimental platform that can support functional testing of individual genetic variants necessary for precision medicine efforts and targeted drug screening/validation. Further, since all tumors including PDXs continue to evolve during serial propagation in vivo, synthetic tumors represent perhaps the only means by which we can explore early events in cellular transformation and segregate their biology from confounding effects of multiple and varied secondary events that accumulate in highly "evolved" samples. Disclosures Steidl: Seattle Genetics: Consultancy; Tioma: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

Download Full-text

Pre-Clinical Efficacy of Co-Targeting GFI1/KDM1A and BRD4 or JAK1/2 Against AML and Post-MPN Secondary AML Blast Progenitor Cells

Blood ◽

10.1182/blood-2020-140212 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-27

Author(s):

Warren Fiskus ◽

Christopher Peter Mill ◽

Christine Birdwell ◽

Bernardo H Lara ◽

Prithviraj Bose ◽

...

Keyword(s):

Progenitor Cells ◽

Research Funding ◽

Target Genes ◽

Amine Oxidase ◽

Colony Growth ◽

Protein Domain ◽

Rna Seq ◽

Protein Levels ◽

Gene Sets ◽

Induced Apoptosis

Transcriptional regulators (TFs) involved in cell-growth, differentiation and survival of AML stem/progenitor cells (LSCs) include RUNX1, PU.1, CEBPα, c-Myb and c-Myc. LSD1 (KDM1A) is an FAD-dependent amine-oxidase that demethylates mono and dimethyl histone H3 lysine 4 (H3K4Me1 and H3K4Me2). LSD1 is part of the repressor complexes involving GFI1, CoREST and HDAC1/2, that regulate active super-enhancers/enhancers (SEs/Es) and their target genes, mediating transcriptional repression and differentiation block in LSCs. GFI1 is a zinc-finger transcriptional repressor involved in AML development and differentiation. GFI1 contains an N-terminal domain through which it binds to the CoREST/LSD1/HDAC1/2 complex to regulate differentiation in LSCs. CRISPR-suppressor scanning revealed that enzymatic activity of LSD1 was not required for LSC differentiation, instead disruption of binding of LSD1 to GFI1 and CoREST induced differentiation in LSCs. LSD1 and GFI1 expression correlates with worse prognosis in MDS/AML. In present studies, we demonstrate first-time ever that knockout (KO) or degradation of LSD1 utilizing CRISPR-Cas9 or LSD1-FKBP12(F36V) and dTAG-13, respectively, disrupted LSD1-binding to GFI1/1B and CoREST, inhibiting colony growth and inducing differentiation markers (CD86 and CD11b) and morphologic differentiation of AML and post-MPN sAML blast progenitor cells (BPCs). CRISPR-mediated knockout of LSD1 in the AML OCI-AML5 and sAML SET2 cells significantly increased the permissive H3K4Me2/3-marked chromatin, reduced H3K27Ac occupancy at SEs/Es (by ChIP-Seq), especially of c-Myc and CDK6, as well as repressed DNMT1, CoREST, c-Myc, CDK6, and c-KIT, while inducing GFI1, PU.1, CEBPα, p21, CD11b, and CD86 levels (log2 -fold change by RNA-Seq and by Western analyses). This correlated with growth inhibition, % differentiation and apoptosis of AML and sAML cells. CRISPR-mediated GFI1-KO ± the irreversible LSD1 inhibitor (LSD1i) (INCB059872, INCB), repressed GFI1 levels, yet enhanced expressions of PU.1, p21 and CD11b and significantly increased % morphologic differentiation. Treatment with INCB (0.25 to 1.0 µM) also disrupted binding of LSD1 to GFI1 and to CoREST, increased GFI1/1B and PU.1 and repressed c-Myc protein levels, while significantly inhibiting colony growth, inducing differentiation and loss of viability of AML and post-MPN sAML (SET2 and HEL92.1.7) cells, as well as patient-derived AML and post-MPN sAML blasts (p < 0.01). Following INCB treatment, ATAC-Seq analysis demonstrated gained peaks in GFI1 and PU.1-target genes. Following H3K27Ac ChIP-seq analysis rank-ordering of SEs (ROSE) plot highlighted active SEs of RUNX1, GFI1, BCL2, PU.1, IRF8 and SMYD3, accompanied by increased H3K27Ac occupancy at the chromatin of GFI1 and PU.1 targets. Notably, INCB treatment also increased BRD4 occupancy, especially at the GFI1 and PU.1-target genes. RNA-Seq analysis showed that INCB treatment perturbed mRNA expressions, with positive normalized enrichment scores (NES) for interferon α, inflammatory-response, GFI1-targets and E2F-target gene-sets, and negative NES for c-Myc-targets and oxidative-phosphorylation gene-sets. RNA-Seq analyses of INCB-treated compared to untreated OCI-AML5 and SET-2 cells also demonstrated log2 fold-increase in the mRNA expressions of GFI1, PU.1 and CEBPα target-genes. Utilizing a protein domain-scanning CRISPR-Cas9 sgRNA screen followed by LSD1i treatment, present studies also demonstrate co-dependencies, including BRD4, in AML cells. BET inhibitor (BETi) treatment also depleted LSD1 protein levels, and co-treatment with the BETi OTX015 and INCB induced synergistic lethality in AML and post-MPN sAML blasts (Combination Indices < 1.0). Pre-treatment with INCB re-sensitized JAKi-resistant sAML cells to ruxolitinib-induced apoptosis and BETi-resistant post-MPN sAML cells to BETi-induced apoptosis. Notably, co-treatment with INCB (1.5 mg/kg) and ruxolitinib (20 mg/kg) or OTX015 (50 mg/kg), administered orally for 21 days, compared to ruxolitinib alone or vehicle control, significantly reduced the sAML burden and improved survival of immune-depleted mice engrafted with luciferized sAML HEL92.1.7 xenografts (p < 0.01). Collectively, these findings support further pre-clinical development of LSD1i-based combinations with ruxolitinib and BETi against post-MPN sAML. Disclosures Bose: CTI BioPharma: Honoraria, Research Funding; NS Pharma: Research Funding; Celgene Corporation: Honoraria, Research Funding; Pfizer, Inc.: Research Funding; Constellation Pharmaceuticals: Research Funding; Astellas Pharmaceuticals: Research Funding; Blueprint Medicines Corporation: Honoraria, Research Funding; Promedior, Inc.: Research Funding; Incyte Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau; Kartos Therapeutics: Honoraria, Research Funding. Kadia:Incyte: Research Funding; Pulmotec: Research Funding; Cellenkos: Research Funding; Celgene: Research Funding; Amgen: Research Funding; Genentech: Honoraria, Research Funding; JAZZ: Honoraria, Research Funding; Cyclacel: Research Funding; Novartis: Honoraria; Ascentage: Research Funding; Astellas: Research Funding; Pfizer: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Astra Zeneca: Research Funding; BMS: Honoraria, Research Funding. Verstovsek:CTI Biopharma Corp: Research Funding; AstraZeneca: Research Funding; Sierra Oncology: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Incyte Corporation: Consultancy, Research Funding; PharmaEssentia: Research Funding; Blueprint Medicines Corp: Research Funding; NS Pharma: Research Funding; Roche: Research Funding; Gilead: Research Funding; Protagonist Therapeutics: Research Funding; Promedior: Research Funding; Genentech: Research Funding; Celgene: Consultancy, Research Funding; ItalPharma: Research Funding.

Download Full-text