Abstract

Associated with approximately 8-13% cases of acute myeloid leukemia (AML), t (8;21) results in the formation of the fusion oncoprotein AML1-ETO (AE), which appears to promote hematopoietic precursor expansion through aberrant regulation of canonical AML1 targets. Multiple lines of evidence indicate AE is insufficient for leukemogenesis and requires cooperative secondary mutations. Interstitial deletions of chromosome 9 are one of the more frequent associated cytogenetic abnormalities seen with t(8;21). We previously identified that knockdown of TLE1 and TLE4, members of the Groucho/TLE family of co-repressors residing in a commonly deleted region of chromosome 9 in t (8;21) del (9q) AML cooperates with AE to drive myeloblastic proliferation and cause a leukemic phenotype. In this study, we show how TLE4 loss in the context of AML1-ETO expression results in cell proliferation, decreased apoptosis, blocked differentiation, and increased resistance to chemotherapy and that these effects are mediated through an upregulation of cyclooxygenase (COX)-dependent inflammation.

TLE4 knockdown via shRNA (shTLE4) in Kasumi-1 cells (derived from a patient with t (8;21) AML) resulted in increased cell cycling and decreased cell death that is concomitant with significantly lower levels of p16Ink4 and p27Kip1 expression, both major regulators of the G1/S cell cycle checkpoint. RNAseq with GSEA analysis identified enrichment of pathways related to IFN signaling and inflammation within upregulated genes in shTLE4 cells compared to control. Filtering for genes with log2 fold change greater than 0.6, we identified three genes related to inflammation (PTGER4, CEBPb, FOS). As demonstration of the relevance of these findings, we showed these expression levels were also higher in primary human del (9q) and t (8;21) del (9q) leukemia samples compared to normal CD34+ cells; revealing a potential leukemogenic role for inflammatory pathways in del (9q) and t (8;21) del (9q) AML. Using a sub-lethal dose of indomethacin (50uM INDM), we found modulating the COX-dependent inflammatory axis decreased growth of shTLE4 Kasumi-1 cells, decreased cell cycling, and increased dead populations. Concomitantly, INDM treatment was able to reverse shTLE4-induced increases in PTGER4, CEBPb, FOS, and IL1b expression to levels similar to those in control cells.

Given the ability to induce myeloblastic proliferation with TLE knockdown and AE expression in zebrafish, we investigated whether shTLE4 causes changes in key myeloid differentiation regulators in Kasumi-1 cells. qPCR analysis demonstrated significantly repressed ELANE, MPO, and PU.1 expression in shTLE4-treated cells, which is relieved with the addition of INDM. TLE4 knockdown in HL60 cells (derived from t (15;17) PML/RARA leukemia) increased levels of FOS, IL1b, CEBPb, and PTGER4. At the same time, shTLE4 significantly reduced CD11b+ and CD11b+ CD14+ populations while decreasing expression of PU.1 and MYB - demonstrating a similar inflammatory gene signature associated with a myeloid differentiation block found in the Kasumi-1 model.

We next wanted to examine whether inhibiting COX-dependent inflammation can be synergistic with existing therapies. INDM significantly increased the cell death and apoptosis seen in both control cells as well as cells with TLE4 knockdown treated with AraC. The loss of TLE4 conferred resistance to combination treatment with indomethacin and AraC as evidenced by increased cell proliferation and decreased cell death. However, we observed that cells with TLE4 knockdown were significantly more sensitive to the combination of sunitinib and INDM, pointing to a potentially more therapeutic and less toxic treatment regimen for this otherwise resistant cell population.

In this study, we have demonstrated that loss of TLE4 appears to contribute to leukemogenesis through differentiation arrest and increased proliferation mediated through upregulation of COX-dependent inflammatory pathways. Our findings suggest that COX inhibition may serve as an effective adjuvant therapy for AML.

Disclosures

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.