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  • br Results br Discussion The advent of TKI


    Discussion The advent of TKI treatment has greatly improved CML therapy. Because CML LSCs are less sensitive to TKIs, CML is usually controlled rather than cured, highlighting an unmet clinical need. Developed from our previous finding that CML LSCs are more dependent on Tcf1 and Lef1 transcription factors than normal HSCs for self-renewal (Yu et al., 2016), this study delves into the concept of using Tcf1/Lef1-dependent genes as a therapeutic target in LSCs (Ashton et al., 2012). Through CMAP data mining, we identified PGE1 and its analog misoprostol as potent agents that simulate key gene FAUC-365 changes, mainly downregulation of Fosb and Fos, caused by Tcf1/Lef1 deficiency in LSCs. PGE1 greatly suppressed LSC activity in a murine CML model and a xenograft model of transplanted human CML LSCs but did not detectably affect normal hematopoiesis. The effect of Tcf1/Lef1 ablation and PGE1 treatment appeared to be specific to CML, because neither affected AML LSC self-renewal or AML progression in a murine MLL-AF9 model (data not shown). The synergistic effect of combining PGE1 and TKI regimens makes PGE1 an ideal therapeutic candidate for treating CML. PGE1 (clinically known as alprostadil) and misoprostol are FDA-approved drugs. PGE1 has a vasodilative effect, which is used to treat erectile dysfunction, pulmonary hypertension, and peripheral artery occlusive disease (Murali et al., 1992, Weiss et al., 2002). Misoprostol is a gastric anti-secretory agent with protective effects on the gastrointestinal mucosa. In this study, PGE1 was used at 2.5–5 mg/kg body weight (equivalent to 7–14 μM), a concentration close to clinically relevant levels at 5–10 μM (Weiss et al., 2004). These considerations suggest that PGE1 and misoprostol can be repositioned from their conventional use to CML treatment, allowing an expedited translational application (Ashburn and Thor, 2004). PGE1 stimulation partly simulated gene expression changes caused by Tcf1/Lef1 deficiency, particularly downregulation of Fosb and Fos. Importantly, PGE1-mediated AP-1 repression was conserved in human CML LSCs in the xenograft model. AP-1 factors are known to regulate cell differentiation, stress response, and tumorigenesis (Lopez-Bergami et al., 2010); however, their roles in HSCs or LSCs have not been extensively investigated (Rossi et al., 2012). We discovered that several AP-1 genes showed strong upregulation in CML LSCs compared with HSPCs in the murine CML model and CML patient samples, and significantly, forced expression of Fosb and Fos in LSCs conferred resistance to PGE1 treatment in the murine CML model. In osteosarcoma and endometrical carcinoma, Fos expression is associated with high-grade lesions and adverse outcome (Bamberger et al., 2001, Gamberi et al., 1998). Identification of Fosb and Fos as key targets to subvert CML LSCs may lead to the discovery of additional therapeutic options, and these factors may be utilized as new biomarkers in assessing therapeutic efficacy and prognosis in CML treatment. In spite of their structural similarity, PGE1 and PGE2 have clearly distinct biological effects. PGE2 has been reported to enhance human cord-blood stem cell xenotransplants (Goessling et al., 2011), partly owing to its ability to activate β-catenin (Goessling et al., 2009). Despite some contentions over the role of β-catenin in normal HSPCs, it is consistently found that β-catenin is required for development and maintenance of LSCs in several leukemias (Heidel et al., 2012, Jamieson et al., 2004, Wang et al., 2010, Yeung et al., 2010, Zhao et al., 2007) and represents a useful drug target in cancer therapy (Anastas and Moon, 2013). COX inhibitors (e.g., indomethacin) are used to block production of E prostanoids—in particular, PGE2—and to prevent β-catenin stabilization in LSCs (Heidel et al., 2012). By direct comparison with PGE1, however, indomethacin showed less profound beneficial effects on CML. PGE1 treatment did not cause β-catenin accumulation or downregulation of Tcf1 or Lef1 expression per se, and PGE1-mediated repression of Fosb and Fos in LSCs was not dependent on β-catenin. Therefore, the action of PGE1 is a simulation of part of gene expression changes caused by genetic ablation of Tcf1 and Lef1 and is not necessarily directly linked to the Tcf/Lef-β-catenin pathway. Further mechanistic analysis showed that PGE1 mainly acted on EP4 receptor and integrated signals from MEK, PI3K, and intracellular Ca2+ to achieve repression of AP-1 factors. Although PGE1 and PGE2 are produced from the same precursors, PGE1 treatment represents a novel and potent approach to subvert CML LSCs, independent of blocking the PGE2-β-catenin pathway.