Key Points• GPR84 simultaneously augments b-catenin signaling and an oncogenic transcription program essential for establishment of MLL.• Our study demonstrates a strong dependence of hematopoietic stem cell-derived MLL leukemic cells on GPR84 for disease maintenance in vivo.b-catenin is required for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). Targeted inhibition of b-catenin signaling has been hampered by the lack of pathway components amenable to pharmacologic manipulation. Here we identified a novel b-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in human and mouse AML LSCs compared with hematopoietic stem cells (HSCs). Suppression of GPR84 significantly inhibited cell growth by inducing G1-phase cell-cycle arrest in pre-LSCs, reduced LSC frequency, and impaired reconstitution of stem cell-derived mixed-lineage leukemia (MLL) AML, which represents an aggressive and drug-resistant subtype of AML. The GPR84-deficient phenotype in established AML could be rescued by expression of constitutively active b-catenin. Furthermore, GPR84 conferred a growth advantage to Hoxa9/Meis1a-transduced stem cells. Microarray analysis demonstrated that GPR84 significantly upregulated a small set of MLL-fusion targets and b-catenin coeffectors, and downregulated a hematopoietic cell-cycle inhibitor. Altogether, our data reveal a previously unrecognized role of GPR84 in maintaining fully developed AML by sustaining aberrant b-catenin signaling in LSCs, and suggest that targeting the oncogenic GPR84/b-catenin signaling axis may represent a novel therapeutic strategy for AML. (Blood. 2014;124(22):3284-3294) IntroductionThe ability for self-renewal, migration/invasion, and drug resistance are fundamental properties of malignant stem cells that drive both disease progression and relapse. Identification of pathways and their molecular components essential for the regulation of abnormally acquired stem cell-like properties is a prerequisite for understanding the underlying mechanisms of oncogenesis and designing effective anticancer therapeutic strategies. We and others have previously shown that mixed-lineage leukemia (MLL) fusion proteins can aberrantly activate Wnt/b-catenin signaling in hematopoietic stem cells (HSCs) or more differentiated granulocyte-macrophage progenitors (GMPs) for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia subtype M5 (AML-M5) 1,2 ; however, the mechanisms involved remain obscure. It has also been noted that activation of b-catenin is observed in tumors without clear mutations in major components of this pathway or an increase in Wnt signaling.3 This suggests that other developmental signaling pathways may be capable of inducing activation or downstream signaling of b-catenin.G protein-coupled receptors (GPCRs), the largest family of cell-surface molecules with key roles in transmitting signals to downstream effectors, ...

Genome-wide studies have identified a high-risk subgroup of pediatric acute lymphoblastic leukemia (ALL) harboring mutations in the Janus kinases (JAKs). The purpose of this study was to assess the preclinical efficacy of the JAK1/2 inhibitor, AZD1480, both as a single agent and in combination with the MEK inhibitor selumetinib, against JAK-mutated patient-derived xenografts. Patient-derived xenografts were established in immune-deficient mice from bone marrow or peripheral blood biopsy specimens, and their gene expression profiles compared with the original patient biopsies by microarray analysis. JAK/STAT and MAPK signaling pathways, and the inhibitory effects of targeted drugs, were interrogated by immunoblotting of phosphoproteins. The anti-leukemic effects of AZD1480 and selumetinib, alone and in combination, were tested against JAK-mutated ALL xenografts both in vitro and in vivo. Xenografts accurately represented the primary disease as determined by gene expression profiling. Cellular phosphoprotein analysis demonstrated that JAK-mutated xenografts exhibited heightened activation status of JAK/STAT and MAPK signaling pathways compared with typical B-cell precursor ALL xenografts, which were inhibited by AZD1480 exposure. However, AZD1480 exhibited modest single-agent in vivo efficacy against JAK-mutated xenografts. Combining AZD1480 with selumetinib resulted in profound synergistic in vitro cell killing, although these results were not translated in vivo despite evidence of target inhibition. Despite validation of target inhibition and the demonstration of profound in vitro synergy between AZD1480 and selumetinib, it is likely that prolonged target inhibition is required to achieve in vivo therapeutic enhancement between JAK and MEK inhibitors in the treatment of JAK-mutated ALL.

The p53 protein is a primary mediator of cellular apoptosis and growth arrest after exposure to DNA-damaging agents. Previous work has shown that the majority of childhood acute lymphoblastic leukemia (ALL) cases express a wild type p53 gene, although the functionality of the p53 pathway has rarely been validated. In the present study, the integrity of the p53 pathway