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. 2016 Sep:48:46-56.
doi: 10.1016/j.leukres.2016.07.002. Epub 2016 Jul 21.

TLE4 regulation of wnt-mediated inflammation underlies its role as a tumor suppressor in myeloid leukemia

Thomas H Shin  1 , Christopher Brynczka  2 , Farshid Dayyani  2 , Miguel N Rivera  3 , David A Sweetser  4
Affiliations

TLE4 regulation of wnt-mediated inflammation underlies its role as a tumor suppressor in myeloid leukemia

Thomas H Shin et al. Leuk Res. 2016 Sep.

Abstract

The presence of AML1-ETO (RUNX1-CBF2T1), a fusion oncoprotein resulting from a t(8;21) chromosomal translocation, has been implicated as a necessary but insufficient event in the development of a subset of acute myeloid leukemias (AML). While AML1-ETO prolongs survival and inhibits differentiation of hematopoietic stem cells (HSC), other contributory events are needed for cell proliferation and leukemogenesis. We have postulated that specific tumor suppressor genes keep the leukemic potential of AML1-ETO in check. In studying del(9q), one of the most common concomitant chromosomal abnormalities with t(8;21), we identified the loss of an apparent tumor suppressor, TLE4, that appears to cooperate with AML1-ETO to confer a leukemic phenotype. This study sought to identify the molecular basis of this cooperation. We show that the loss of TLE4 confers proliferative advantage to leukemic cells, simultaneous with an upregulation of a pro- inflammatory signature mediated through aberrant increases in Wnt signaling activity. We further demonstrate that inhibition of cyclooxygenase (COX) activity partly reverses the pro-leukemic phenotype due to TLE4 knockdown, pointing towards a novel therapeutic approach for myeloid leukemia.

Keywords: AML1-ETO; Acute myeloid leukemia; Inflammation; TLE4; Tumor suppressor; Wnt signaling.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
Kasumi-1 proliferation, apoptosis, and differentiation are sensitive to TLE4 expression levels. (A) qRT-PCR using RNA from Kasumi-1 cells treated with two TLE4-specific shRNA confirms knockdown of TLE4 message by at least 60% (n = 3 biologic triplicates with technical triplicates. *: p < 0.05). Western blot confirms over 75% decrease in TLE4 protein in Kasumi-1 and 293T cells treated with shTLE4 1. (B) Cell count of GFP+ Kasumi-1 cells treated with scramble control or two unique TLE4-specific shRNA coexpressed with IRES-GFP in lentivirus were tracked over 18 days. (C) Similarly treated cells were fixed and stained with for DAPI cell cycle or (D) stained for Annexin V analysis 7 days post-lentiviral infection. Dead population was defined as Annexin V+ and DAPI+ cells. (E) qRT-PCR query of two repressive targets of AML1- ETO, CEBPb and GATA1, reveal decreased levels of expression in TLE4 knockdown Kasumi-1 cells. (All experiments carried out in biologic triplicates and repeated at least twice with technical triplicates. *: p < 0.05, **: p < 0.01, ***: p < 0.001).
Fig. 2
Fig. 2
TLE4-mediated increases in inflammatory gene expression are concomitant with inhibition of pharmacologically induced myeloid differentiation in Kasumi-1 and HL60 cells. (A) Flow cytometry shows percentage of control and T4KD GFP+ Kasumi-1 cells that are CD14+ after culture in media supplemented with DMSO or 10 uM ATRA. (B) Flow cytometry analysis shows blunted induction of CD14+ populations in shTLE4-treated HL60 cells when cultured in media supplemented with 0.1 uM vitamin D3. (C) RNA harvested from shTLE4 or control shRNA treated HL60 cells were used in qRT-PCR seven days post- spinoculation to query for myeloid transcription factors. (All experiments carried out in biologic and technical triplicates. *: p < 0.05, ***: p < 0.001, ****: p < 0.0001).
Fig. 3
Fig. 3
RNAseq identifies enrichment of immune system and inflammatory response pathways in genes upregulated in T4KD Kasumi-1 cells seven days post-spinoculation. (A) Heatmap summarizing differentially expressed genes between T4KD and control Kasumi-1 cells (n = 2 biologic replicates, filtered for FDR <0.2). (B) qRT-PCR verification of select upregulated genes with FDR <0.2 and log2 fold change >0.6 identified from RNAseq (n = 3 biologic replicates with technical triplicates, *: p < 0.05). (C) GSEA plots from analysis identifying pathways enriched in upregulated geneset (p and q values obtained from GSEA analysis).
Fig. 4
Fig. 4
Elevated expression of inflammatory genes is characteristic of t(8;21) AML and is seen concomitant with increased Wnt signaling in AML cell lines with T4KD. (A) qRT-PCR query of CEBPb, PTGER4, and FOS using RNA harvested from primary human bone marrow samples reveal similar increases in inflammatory gene expression in del(9q) and t(8;21) del(9q) AML versus healthy CD34+ cells. qRT-PCR using RNA harvested from (B) Kasumi-1 cells and (C) HL60 cells seven days after lentiviral T4KD reveals increases in COX1, COX2, and inflammatory genes related to prostaglandin metabolism and downstream mediators. (A: n = 10���12 biologic replicates with technical triplicates per arm. B–C: n = 3 biologic replicates with technical triplicates. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).
Fig. 5
Fig. 5
Wnt signaling in the context of AML1-ETO is sensitive to TLE4 levels. (A) qRT-PCR with probes for inflammatory genes identified by RNAseq and other Wnt targets was performed using RNA harvested from Kasumi-1 cells cultured in media supplemented with either 10 nM recombinant human Wnt3a or DMSO. (B) qRT-PCR using RNA from AML1-ETO expressing 293T cells shows significant increases in COX1 and COX2 expression compared to control naive 293T cells (C) TOP/FOP ratios calculated from firefly and renilla luciferase activity of AML1 -ETO expressing 293T cells co-transfected with TOPFlash/FOPFlash. Presence of AML1-ETO increases Wnt signaling activity. Addition of TLE4 expression vector is able to abrogate AE-induced Wnt signaling, consistent with other studies confirming role of TLE4 as a regulator of Wnt signaling.
Fig. 6
Fig. 6
COX inhibitor INDM is able to modulate T4KD- dependent Wnt signaling in AML1-ETO expressing cells. (A) TOP/FOP ratios were calculated using fire-fly and renilla luciferase activity of 293T cells 48 h after nucleoporation with TOPFlash/FOPFlash reporter constructs, AML1-ETO expression vector, and either control or TLE4-specific shRNA. Assay reveals increased Wnt signaling activity in T4KD 293T cells compared to control, which is inhibited by addition of 50 uM indomethacin in culture media. (B) qRT-PCR analysis of cells used in (A) reveals significant increases in COX1 and COX2 expression due to T4KD and subsequent blunting with INDM treatment (All experiments carried out in biologic triplicates with technical triplicates for all experiments. *: p < 0.05, **: p < 0.01, ***: p < 0.001).
Fig. 7
Fig. 7
Wnt inhibitor ICG-001 is able to suppress T4KD-induced cell growth and expression of inflammatory genes and Wnt targets. (A) Kasumi-1 cells treated with lentiviral TLE4-specific or control shRNA delivery were tracked for fold change in GFP+ cells in the presence of either 10 nM ICG-001 or DMSO. While T4KD confers increased cell proliferation, ICG-001 is able to stunt growth of Kasumi-1 cells regardless of TLE4 status (n = 3 biologic replicates; statistics shown for comparison of growth rates to Control + DMSO arm). (B) RNA from aforementioned cells was harvested at day 15 of culture for qRT-PCR. Assays reveal ICG-001 is able to block T4KD-induced increases in inflammatory and Wnt target gene expression. (n = 3 biologic triplicates with technical triplicates. *: p < 0.05, **: p < 0.01, ***: p < 0.001).
Fig. 8
Fig. 8
Indomethacin is able to reverse cell proliferation and drug resistance due to T4KD in Kasumi-1 cells. (A) T4KD and control Kasumi-1 cells were tracked for fold change in GFP+ cells in the presence of either 50 uM INDM or DMSO. Presence of INDM is able to stunt T4KD-induced cell growth (n = 3 biologic replicates; statistics shown for comparison of growth rates to Control + DMSO arm). (B) T4KD and control Kasumi-1 cells were cultured in DMSO, 50 uM INDM, 100 uM araC, or combination of 50 uM INDM and 100 uM araC. After seven days of treatment, cells were stained for Annexin V analysis and checked for dead GFP+ cell populations, which revealed INDM is able to reverse T4KD-induced resistance to araC treatment. Dead population was defined as Annexin V+ and DAPI+ cells. (n = 3 biologic replicates with technical triplicates. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).
Fig. 9
Fig. 9
COX inhibition is able to partially reverse T4KD-mediated induction of inflammatory genes in Kasumi-1 and AML1-ETO-expressing 293T cells. qRT-PCR was performed using RNA harvested from either Kasumi-1 or AML1-ETO- expressing 293T cells cultured in media supplemented with either 50 uM indomethacin or DMSO. Query reaffirms induction of inflammatory genes with T4KD, which is abrogated in cells cultured in indomethacin. (n = 3 biologic replicates with technical triplicates. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).
Fig. 10
Fig. 10
T4KD-induced suppression of myeloid transcription factors is reversible by COX inhibition. RNA for qRT-PCR was harvested from Kasumi-1 cells treated with 50 uM INDM or DMSO after lentiviral delivery of TLE4 or control shRNA. Assays reveal T4KD-induced suppression of myeloid transcription factors is relieved when cells were cultured in 50 uM INDM. (n = 3 biologic replicates with technical triplicates. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).
Fig. 11
Fig. 11
Schematic diagram summarizing proposed TLE4 regulation of AML1-ETO/COX/Wnt axis. TLE4 regulates AML1-ETO-mediated inflammatory signature by functioning as an “engine break”-like repressor of Wnt signaling. Blockade of the COX-Wnt signaling axis by Wnt inhibitor ICG-001 or COX inhibitor indomethacin is able to abrogate T4KD-induced pro-leukemic effects in t(8;21) leukemia cells. Direct interactions between TLE4 and AML1-ETO may provide an additional level of regulation.
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