Highlights d RSPO-LGR4 is essential for stem cell self-renewal in a subset of AML d LGR4 promotes aberrant self-renewal through cooperation with HOXA9 d RSPO3 acts as a stem cell growth factor to sustain proliferation of AML patient blasts d Clinical-grade anti-RSPO3 impairs LSC self-renewal in patient-derived xenografts
Although the clinical importance of aberrant Wnt/β-catenin signaling has been recognized in various cancers, including MLL-rearranged acute myeloid leukemia (MLL AML), its key tractable pathway components have not yet been discovered in leukemic stem cells (LSC). Our studies have identified an Rspo3/Wnt3a-Lgr4-Gnaq pathway, which significantly potentiates β-catenin signaling in MLL LSC. Genetic and pharmacological targeting of this pathway impairs LSC self-renewal and survival, inhibiting MLL-AF9-induced leukemia progression in vivo. Gene expression analysis of AML patient samples (Nucleic Acids Res, 41:D1034-9, 2013) revealed an approximately 3-fold increase (p=0.00002) in expression of leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) in leukemic cells from patients with MLL AML compared to normal human hematopoietic stem cells (HSC). As recent studies have highlighted a critical link between R-spondin (Rspo)/Lgr4 and Wnt/β-catenin signaling pathways, we hypothesized that up-regulation of Lgr4 is associated with aberrant activation of β-catenin signaling in MLL LSC. We have previously demonstrated that β-catenin is highly expressed in HSC transformed by MLL-AF9 and is lower in HSC transduced with leukemic oncogenes such as Hoxa9/Meis1, while increased β-catenin expression is correlated with a poor survival rate in mice. In this study, western blots confirmed high levels of Lgr4 expression in HSC expressing MLL-AF9 compared to Hoxa9/Meis1. ShRNA-mediated stable knockdown of Lgr4 markedly reduced colony formation of HSC expressing MLL-AF9 by 55-65% (p=0.0001) and significantly prolonged mouse survival (p=0.0019) through its inhibition of endogenous β-catenin expression. This deficient phenotype could be rescued by expression of a constitutively active form of β-catenin. Furthermore, ectopic expression of Lgr4 alone was not sufficient for triggering the leukemic transformation of HSC but conferred a growth advantage in vivo to HSC expressing Hoxa9/Meis1 and significantly accelerated the onset of Hoxa9/Meis1-induced AML in mice (p=0.0011). These data support an oncogenic role of Lgr4 in promoting tumor formation through activation of β-catenin signaling. As Lgr4 has recently been identified as a receptor for the Rspo family of secreted proteins (Rspo1–Rspo4), we sought to determine if Rspo is a positive regulator of β-catenin signaling in MLL AML. We found that only the combination of Rspo3 and Wnt3a potently enhanced β-catenin signaling in HSC expressing MLL-AF9 whereas Rspo and Wnt3a alone or the combination of Wnt3a with other Rspo had no effects on β-catenin activity. Depletion of Lgr4 completely abolished Rspo3/Wnt3a-induced β-catenin signaling, suggesting Rspo3/Wnt3a potentiating β-catenin signaling through Lgr4. Next, we assessed if Lgr4 signals through G protein pathways. By testing G protein alpha inhibitors in MLL LSC, we demonstrated that G protein alpha-q (Gnaq) was required for maintenance of stem cell properties by chemical suppression of the Gnaq-activated β-catenin pathway with a Gnaq selective inhibitor, which exhibited a 3-fold decrease in colony formation (p=0.0001) and a 4-fold reduction in cell number (p=0.0009), and was sufficient to induce substantial cell differentiation and apoptosis. Treatment with Gnaq inhibitor abolished the effect of Lgr4 on β-catenin transactivation, implicating an Lgr4-Gnaq-β-catenin signaling pathway in MLL LSC. Microarray analysis of gene expression confirmed enrichment of genes related to cancer cell proliferation, migration and growth, as well as enrichment of Wnt target genes in LSC expressing Lgr4. Taken together, we report here an Rspo3/Wnt3a-Lgr4-Gnaq-β-catenin signaling circuit in MLL leukemogenesis. Interference with components of the circuit can block β-catenin signaling and perturb leukemia development. Thus, our findings provide potential therapeutic targets in treating LSC-based hematological malignancy driven by Wnt/β-catenin signaling. Disclosures: No relevant conflicts of interest to declare.
Acute myeloid leukemia (AML) is a lethal blood cancer. Clinical evidence has highlighted the critical role for leukemic stem cells (LSCs) in the high relapse rate of AML patients. Understanding the signaling pathways critical for LSC function will facilitate the development of new therapies to target LSCs. G protein-coupled receptors (GPCRs) are the most successful drug targets with approximately 36% of currently marketed drugs targeting human GPCRs (Rask-Andersen et al., Nat Rev Drug Discov 2011). Aberrant expression of GPCRs has been observed in various cancers and the importance of GPCRs in cancer stem cells has begun to be appreciated. In this study, our gene expression profiling analysis identified a novel GPCR (hereafter named NG), which was suppressed in LSCs compared to normal hematopoietic stem cells. Overexpression of NG in pre-LSCs severely impaired leukemia initiation and progression in mice, whereas knockdown of NG significantly accelerated the disease onset. Our data also showed that NG overexpression substantially downregulated several well-known Wnt/β-catenin targets (e.g., Tcf7l2, c-Fos and Ccnd1). These data support a tumor suppressive role for NG in inhibiting leukemogenesis via downregulation of Wnt/β-catenin signaling. Consistent with these observations, treatment with an agonist that specifically activates the NG signaling pathway induced a marked decrease in cell viability and growth in murine LSCs and in human AML THP-1 cells, but had no effects on human AML MOLM-13 cells. This agrees with our finding that NG specifically targets the Wnt/β-catenin signaling, and the inhibitory effect of the agonist might largely depend on β-catenin expression levels in human AML cells, wherein THP-1 has been reported to express endogenous β-catenin but MOLM-13 exhibits no detectable β-catenin expression (Man CH. et al. Blood 2015). In summary, our data support a tumour suppressor role for NG in leukemogenesis and the forced expression of NG may provide a means to eradicate LSCs. Thus, restoring NG by the agonist treatment represents a promising therapeutic strategy for AML treatment. Citation Format: Florida Voli, Hangyu Yi, Estrella Gonzales-Aloy, Jenny Yingzi Wang. Targeting a novel G-protein coupled receptor (GPCR) for elimination of leukemia stem cells (LSC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3906. doi:10.1158/1538-7445.AM2017-3906
Gadd45a is a mediator of growth arrest and its expression is suppressed in primary human MLL-rearranged AML (predominantly MLL-AF9) and AML1-ETO AML with increased FLT3 expression (Valk et al., N Engl J Med 2004; Perugini et al., Leukemia 2009). While Gadd45a is frequently silenced in several human cancers, including leukemia, little is know about how Gadd45a loss contributes to leukemogenesis. Our studies have shown that Gadd45a loss promotes MLL-AF9 mediated development in AML in mice and protects leukemic cells from apoptosis via novel β-catenin/mitochondria pathway. To explore the functional role of Gadd45a loss in MLL-rearranged AML, we retrovirally introduced MLL-AF9 into c-Kithigh progenitors isolated from Gadd45a knockout mouse bone marrow cells. Our results showed that Gadd45a knockout markedly reduced the colony forming capacity of pre-leukemic cells and significantly shortened the latency for MLL leukemia development in mice (p = 0.037, N = 6 mice). As Gadd45a could induce cell death through the mitochondria pathway (Tong et al., Mol Cell Biol 2005), we next examined the expression of proteins involved in the mitochondrial apoptosis in Gadd45a knockout pre-leukemic cells. Our western blot analysis showed that Gadd45a knockout dramatically decreased expression of pro-death proteins Txnip, Bim and Bax but interestingly, significantly increased active β-catenin expression in mitochondria. Given that Gadd45a has been reported to regulate β-catenin distribution (Ji et al., Oncogene 2007), we postulate that Gadd45a loss facilitates the translocation of β-catenin into mitochondria. We have previously demonstrated that β-catenin is highly expressed in MLL-AF9 transformed pre-leukemic cells and is lower in Hoxa9/Meis1 transformed pre-leukemic cells, while increased β-catenin expression is correlated with a poor survival rate in mice. Here, we retrovirally introduced a constitutively active form of β-catenin into Hoxa9/Meis1 pre-leukemic cells. As expected, activation of β-catenin reduced endogenous expression of Txnip and Bim in mitochondria, exhibiting an expression pattern similar to that regulated by Gadd45a knockout. Consistently, both Gadd45a knockout and β-catenin activation not only reduced the basal cellular reactive oxygen species (ROS) levels but also inhibited Antimycin A (an ROS generator)-induced mitochondrial ROS production. Taken together, our study discovers a previously unrecognized Gadd45a loss/β-catenin/mitochondria pathway that prevents mitochondrial ROS accumulation and protects leukemic cells from cell death in MLL leukemogenesis. Disclosures No relevant conflicts of interest to declare.
Acute myeloid leukemia (AML) is still a deadly form of leukemia due to frequent relapse caused by the persistence of drug-resistant leukemic stem cells (LSCs). We have previously demonstrated a crucial role for β-catenin signaling in regulating LSCs and identified GPR84 as an important β-catenin regulator in the maintenance of mixed-lineage leukemia (MLL) LSCs (Wang et al., Science 2010; Dietrich et al., Blood 2014). Hence, targeting LSCs by pharmacological inhibition of GPR84/β-catenin signaling represents a promising therapeutic approach. In collaboration with a pharmaceutical company that has developed a novel GPR84 antagonist (GP), we investigated the effect of GP in MLL pre-leukemic stem cell (pre-LSC) function. GP (20 μM) significantly inhibited the colony forming ability of MLL pre-LSCs (P < 0.0001) but had little effect on normal hematopoietic stem cells. Quantitative RT-PCR and western blot analysis confirmed GP-induced downregulation of GPR84 target genes, including Hoxa5, Hoxa7 and Meis1a, indicating GP-induced inhibition of GPR84 signaling. To further examine the mechanism of GPR84 inhibition on MLL pre-LSCs, we evaluated several epigenetic regulators (i.e. JMJD1c and EZH2) known to promote leukemogenesis (Zhu et al., J Clin Invest 2016; Tanaka et al., Blood 2012). Western blot analysis showed that inhibition of GPR84 signaling did not alter the expression of JMJD1c or EZH2. However, we observed a significant increase in the expression of a novel and not-yet-characterized histone demethylase (HD) in AML. To investigate the role of HD in AML leukemogenesis, we overexpressed HD in MLL pre-LSCs and subsequent serial replating assay showed a marked reduction in colony forming ability (P < 0.005), indicating impaired self-renewal in vitro. Consistent with our in vitro observations, in vivo transplantation in syngeneic mice revealed a significant delay in leukemia onset and increase in mouse survival (P < 0.001). We next performed western blot analysis to examine the demethylase activity of HD, and our data revealed that HD overexpression caused a substantial reduction in global histone 3 lysine 36 dimethylation (H3K36me2), an epigenetic mark normally associated with transcriptional activation and elongation. In order to identify genes regulated by HD through demethylation of H3K36me2, we performed H3K36me2 ChIP-seq on HD overexpressing MLL pre-LSCs. Our analysis identified several genes including anti-apoptotic protein Mcl-1 and angiogenic receptor Nrp1, which are known to be involved in AML leukemogenesis, with decreased H3K36me2 mark on both the transcriptional start site and gene body. Subsequent western blot analysis confirmed the decreased expression of both Mcl-1 and Nrp1 in HD overexpressing pre-LSCs. Given the prominent roles of anti-apoptosis and angiogenesis in the development of hematologic malignancies such as leukemia, we are currently evaluating these mechanisms caused by HD overexpression in an important subtype of AML. Taken together, our study identifies a novel histone demethylase that acts downstream of GPR84 signaling to function as a potent tumor suppressor in the development of MLL LSCs. Disclosures No relevant conflicts of interest to declare.
G-protein coupled receptors (GPCRs) are the most successful drug targets with 36% of currently marketed drugs targeting human GPCRs. F2r, a GPCR that is overexpressed in various human cancers (Ribeiro et al., Oncol Rep 2009), is a positive regulator of the β-catenin pathway and an inhibitor of the JNK pathway in mammalian cells (Sun et al., Nat Cell Biol 2001). The expression of F2r is significantly elevated in aggressive leukemias including blast phase of CML and AML (Veiga et al., Blood Cells Mol Dis 2011). While these observations implicate an involvement of F2r in human leukemia, its function in AML remains unknown. Our preliminary data showed that shRNA-mediated knockdown of F2r markedly decreased active β-catenin in MLL-AF9 mediated pre-leukemia stem cells (pre-LSCs) and leukemia stem cells (LSCs), confirming that F2r is a positive regulator of β-catenin. F2r deficiency decreased LSC colony formation (p=0.0002) in a serial replating assay, indicating a reduction in self-renewal capacity. Furthermore, LSCs exhibited a significant enhancement in apoptotic activity in response to F2r deficiency, displaying a 12-fold increase in apoptosis. Our microarray expression analysis revealed that F2r inhibition significantly reduced the expression of several genes responsible for maintenance of mitochondrial integrity and energy metabolism (mtND4L (p=0.0013), mtND2 (p<0.0001) and mtCytB (p<0.0001)). To investigate this further we used a mitochondria-specific fluorogenic probe to measure reactive oxygen species (ROS) production. A significant increase in ROS production (p=0.0003) indicated that F2r inhibition destabilizes the mitochondrial membrane. This was accompanied by a marked increase, as observed by Western blot analysis, in the proapoptotic proteins Bcl-2-interacting mediator of cell death (Bim) and thioredoxin-interacting protein (Txnip) which permeabilize the mitochondrial membrane releasing cytochrome c and inducing apoptosis. Through oxidative phosphorylation, mitochondria play an essential role in the supply of metabolic energy (ATP) to the cell. F2r deficient LSCs had a significantly reduced rate of oxygen consumption measured using a phosphorescent oxygen probe (p=0.0066) and a significantly lower concentration of basal ATP (p=0.012) compared to control LSCs. F2r inhibition, therefore, induces substantial oxidative stress which triggers the intrinsic apoptotic pathway. To assess the therapeutic value of F2r inhibition, we used the selective non-peptide F2r inhibitor SCH79797. Alamar blue-based cell viability assays showed that SCH79797 was potent against LSCs and had no cytotoxic effects on lineage-negative normal mouse bone marrow cells. F2r inhibitor treatment resulted in a 2-fold reduction in colony forming ability, 3.8-fold enhanced ROS production and inhibited β-catenin activity. BrdU labeling revealed a significant reduction in in vivo short-term proliferation of LSCs that were pre-treated with SCH79797 for 48 hours in culture, transplanted into recipient mice and collected from bone marrow 8 days post-transplantation (p=0.0008). Additional in vivo studies using a mouse model of MLL AML are currently ongoing to further evaluate the therapeutic potential of F2r inhibition. Collectively, our findings suggest that F2r inhibition selectively targets LSC self-renewal, identifying a therapeutic window to eliminate LSCs while preserving normal blood cells. Previous studies suggest that F2r knockdown not only suppresses β-catenin but also activates JNK signaling. Consistently, our Western blot analysis revealed activation of JNK in response to inhibition of F2r. Sustained JNK activation has been reported in many types of AML cells and promotes survival signals during leukemia development (Hess et al., Nat Genet 2002). This suggests that JNK and F2r inhibition could be used in combination to impair LSC self-renewal, with a concurrent increase in cell death. In support of this hypothesis, we have showed that co-inhibition of F2r and JNK induced a potent anti-LSC effect, significantly increasing cell death and ROS production compared to single treatment. The efficacy of this co-treatment is currently being evaluated in primary human AML patient samples in addition to our in vivo mouse model system. Altogether our data suggest a novel LSC-eliminating treatment strategy targeting F2r/β-catenin/JNK signaling for aggressive AML. Disclosures No relevant conflicts of interest to declare.
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