Maintenance and pharmacologic targeting of ROR1 protein levels via UHRF1 in t(1;19) pre-B-ALL.

Expression of the transmembrane pseudokinase ROR1 is required for survival of t(1;19)-pre-B-cell acute lymphoblastic leukemia (t(1;19) pre-B-ALL), chronic lymphocytic leukemia, and many solid tumors. However, targeting ROR1 with small-molecules has been challenging due to the absence of ROR1 kinase activity. To identify genes that regulate ROR1 expression and may, therefore, serve as surrogate drug targets, we employed an siRNA screening approach and determined that the epigenetic regulator and E3 ubiquitin ligase, UHRF1, is required for t(1;19) pre-B-ALL cell viability in a ROR1-dependent manner. Upon UHRF1 silencing, ROR1 protein is reduced without altering ROR1 mRNA, and ectopically expressed UHRF1 is sufficient to increase ROR1 levels. Additionally, proteasome inhibition rescues loss of ROR1 protein after UHRF1 silencing, suggesting a role for the proteasome in the UHRF1-ROR1 axis. Finally, we show that ROR1-positive cells are twice as sensitive to the UHRF1-targeting drug, naphthazarin, and undergo increased apoptosis compared to ROR1-negative cells. Naphthazarin elicits reduced expression of UHRF1 and ROR1, and combination of naphthazarin with inhibitors of pre-B cell receptor signaling results in further reduction of cell survival compared with either inhibitor alone. Therefore, our work reveals a mechanism by which UHRF1 stabilizes ROR1, suggesting a potential targeting strategy to inhibit ROR1 in t(1;19) pre-B-ALL and other malignancies.

The Colony-Stimulating Factor 3 Receptor T640N Mutation Is Oncogenic, Sensitive to JAK Inhibition, and Mimics T618I.

PURPOSE: Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in the majority of chronic neutrophilic leukemia (CNL) and a smaller percentage of atypical chronic myeloid leukemia (aCML) cases. Although CSF3R point mutations (e.g., T618I) are emerging as key players in CNL/aCML, the significance of rarer CSF3R mutations is unknown. In this study, we assess the importance of the CSF3R T640N mutation as a marker of CNL/aCML and potential therapeutic target. EXPERIMENTAL DESIGN: Sanger sequencing of leukemia samples was performed to identify CSF3R mutations in CNL and aCML. The oncogenicity of the CSF3R T640N mutation relative to the T618I mutation was assessed by cytokine independent growth assays and by mouse bone marrow transplant. Receptor dimerization and O-glycosylation of the mutants was assessed by Western blot, and JAK inhibitor sensitivity was assessed by colony assay. RESULTS: Here, we identify a CSF3R T640N mutation in two patients with CNL/aCML, one of whom was originally diagnosed with MDS and acquired the T640N mutation upon evolution of disease to aCML. The T640N mutation is oncogenic in cellular transformation assays and an in vivo mouse bone marrow transplantation model. It exhibits many similar phenotypic features to T618I, including ligand independence and altered patterns of O-glycosylation--despite the transmembrane location of T640 preventing access by GalNAc transferase enzymes. Cells transformed by the T640N mutation are sensitive to JAK kinase inhibition to a similar degree as cells transformed by CSF3R T618I. CONCLUSIONS: Because of its similarities to CSF3R T618I, the T640N mutation likely has diagnostic and therapeutic relevance in CNL/aCML.

Human cytomegalovirus-encoded pUL7 is a novel CEACAM1-like molecule responsible for promotion of angiogenesis.

UNLABELLED: Persistent human cytomegalovirus (HCMV) infection has been linked to several diseases, including atherosclerosis, transplant vascular sclerosis (TVS), restenosis, and glioblastoma. We have previously shown that factors secreted from HCMV-infected cells induce angiogenesis and that this process is due, at least in part, to increased secretion of interleukin-6 (IL-6). In order to identify the HCMV gene(s) responsible for angiogenesis promotion, we constructed a large panel of replication-competent HCMV recombinants. One HCMV recombinant deleted for UL1 to UL10 was unable to induce secretion of factors necessary for angiogenesis. Fine mapping using additional HCMV recombinants identified UL7 as a viral gene required for production of angiogenic factors from HCMV-infected cells. Transient expression of pUL7 induced phosphorylation of STAT3 and ERK1/2 MAP kinases and production of proangiogenic factors, including IL-6. Addition of recombinant pUL7 to cells was sufficient for angiogenesis and was again associated with increased IL-6 expression. Analysis of the UL7 structure revealed a conserved domain similar to the immunoglobulin superfamily domain and related to the N-terminal V-like domain of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Our report therefore identifies UL7 as a novel HCMV-encoded molecule that is both structurally and functionally related to cellular CEACAM1, a proangiogenic factor highly expressed during vasculogenesis. IMPORTANCE: A hallmark of cytomegalovirus (CMV) infection is its ability to modulate the host cellular machinery, resulting in the secretion of factors associated with long-term diseases such as vascular disorders and cancer. We previously demonstrated that HCMV infection alters the types and quantities of bioactive proteins released from cells (designated the HCMV secretome) that are involved in the promotion of angiogenesis and wound healing. A key proangiogenic and antiapoptotic factor identified from a proteomic-based approach was IL-6. In the present report, we show for the first time that HCMV UL7 encodes a soluble molecule that is a structural and functional homologue of the CEACAM1 proangiogenic cellular factor. This report thereby identifies a critical component of the HCMV secretome that may be responsible, at least in part, for the vascular dysregulation associated with persistent HCMV infection.

Ligand independence of the T618I mutation in the colony-stimulating factor 3 receptor (CSF3R) protein results from loss of O-linked glycosylation and increased receptor dimerization.

Mutations in the CSF3 granulocyte colony-stimulating factor receptor CSF3R have recently been found in a large percentage of patients with chronic neutrophilic leukemia and, more rarely, in other types of leukemia. These CSF3R mutations fall into two distinct categories: membrane-proximal mutations and truncation mutations. Although both classes of mutation have exhibited the capacity for cellular transformation, several aspects of this transformation, including the kinetics, the requirement for ligand, and the dysregulation of downstream signaling pathways, have all been shown to be discrepant between the mutation types, suggesting distinct mechanisms of activation. CSF3R truncation mutations induce overexpression and ligand hypersensitivity of the receptor, likely because of the removal of motifs necessary for endocytosis and degradation. In contrast, little is known about the mechanism of activation of membrane-proximal mutations, which are much more commonly observed in chronic neutrophilic leukemia. In contrast with CSF3R truncation mutations, membrane-proximal mutations do not exhibit overexpression and are capable of signaling in the absence of ligand. We show that the Thr-615 and Thr-618 sites of membrane-proximal mutations are part of an O-linked glycosylation cluster. Mutation at these sites prevents O-glycosylation of CSF3R and increases receptor dimerization. This increased dimerization explains the ligand-independent activation of CSF3R membrane-proximal mutations. Cytokine receptor activation through loss of O-glycosylation represents a novel avenue of aberrant signaling. Finally, the combination of the CSF3R membrane proximal and truncation mutations, as has been reported in some patients, leads to enhanced cellular transformation when compared with either mutation alone, underscoring their distinct mechanisms of action.

The CSF3R T618I mutation causes a lethal neutrophilic neoplasia in mice that is responsive to therapeutic JAK inhibition.

We have recently identified targetable mutations in CSF3R (GCSFR) in 60% of chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL-negative) chronic myeloid leukemia (aCML) patients. Here we demonstrate that the most prevalent, activating mutation, CSF3R T618I, is sufficient to drive a lethal myeloproliferative disorder in a murine bone marrow transplantation model. Mice transplanted with CSF3R T618I-expressing hematopoietic cells developed a myeloproliferative disorder characterized by overproduction of granulocytes and granulocytic infiltration of the spleen and liver, which was uniformly fatal. Treatment with the JAK1/2 inhibitor ruxolitinib lowered the white blood count and reduced spleen weight. This demonstrates that activating mutations in CSF3R are sufficient to drive a myeloproliferative disorder resembling aCML and CNL that is sensitive to pharmacologic JAK inhibition. This murine model is an excellent tool for the further study of neutrophilic myeloproliferative neoplasms and implicates the clinical use of JAK inhibitors for this disease.