T618I CSF3R mutations in chronic neutrophilic leukemia induce oncogenic signals through aberrant trafficking and constitutive phosphorylation of the O-glycosylated receptor form.

Activating mutations in the membrane-proximal region of the colony-stimulating factor 3 receptor (CSF3R) are a hallmark of chronic neutrophilic leukemia (CNL) with the T618I mutation being most common. The mechanisms underlying constitutive activation of the T618I CSF3R and its signal propagation are poorly understood. Ligand-independent activation of the T618I CSF3R has previously been attributed to loss of receptor O-glycosylation and increased receptor dimerization. Here, we show that the T618I CSF3R is indeed glycosylated but undergoes enhanced spontaneous internalization and degradation that results in a marked decrease in its surface expression. Inhibition of the proteasome dramatically increases expression of the O-glycosylated T618I CSF3R. We also demonstrate that the O-glycosylated wild-type CSF3R is tyrosine phosphorylated in response to ligand but constitutively phosphorylated in cells expressing T618I CSF3R. Constitutive tyrosine phosphorylation of the O-glycosylated T618I receptor form correlated with activation of JAK2 and both the mutant receptor and JAK2 were found to be constitutively ubiquitinated. These observations provide novel insights into the mechanisms of oncogenic signaling by T618I CSF3R mutations in CNL.

Altered expression of CSF3R splice variants impacts signal response and is associated with SRSF2 mutations.

Three annotated CSF3R mRNA splice variants have been described. CSF3R-V1 is the wild-type receptor, while CSF3R-V4 is a truncated form increased in some patients with AML. CSF3R-V3 mRNA was identified in placenta more than 20 years ago, but remains largely uncharacterized due to the lack of a suitable detection assay. Using a novel digital PCR method to quantitate expression of each CSF3R mRNA splice variant in hematopoietic cells, CSF3R-V1 was most highly expressed followed by CSF3R-V3. Functional assays revealed expression of V3 alone conferred a hypoproliferative phenotype associated with defective JAK-STAT activation. However, coexpression of V1 with V3 rescued proliferative responses. Comparative analysis of V3/V1 expression in CD34+ cells from healthy donors and patients with AML revealed a statistically significant increase in the V3/V1 ratio only in the subset of patients with AML harboring SRSF2 mutations. Knockout of SRFS2 in KG-1 and normal CD34+ cells decreased the V3/V1 ratio. Collectively, these data are the first to demonstrate expression of the CSF3R-V3 splice variant in primary human myeloid cells and a role for SRSF2 in modulating CSF3R splicing. Our findings provide confirmatory evidence that CSF3R is a target of SRSF2 mutations, which has implications for novel treatment strategies for SRSF2-mutated myeloid malignancies.

Bioflavonoids promote stable translocations between MLL-AF9 breakpoint cluster regions independent of normal chromosomal context: Model system to screen environmental risks.

Infant acute leukemias are aggressive and characterized by rapid onset after birth. The majority harbor translocations involving the MLL gene with AF9 as one of its most common fusion partners. MLL and AF9 loci contain breakpoint cluster regions (bcrs) with sequences hypothesized to be targets of topoisomerase II inhibitors that promote translocation formation. Overlap of MLL bcr sequences associated with both infant acute leukemia and therapy-related leukemia following exposure to the topoisomerase II inhibitor etoposide led to the hypothesis that exposure during pregnancy to biochemically similar compounds may promote infant acute leukemia. We established a reporter system to systematically quantitate and stratify the potential for such compounds to promote chromosomal translocations between the MLL and AF9 bcrs analogous to those in infant leukemia. We show bioflavonoids genistein and quercetin most biochemically similar to etoposide have a strong association with MLL-AF9 bcr translocations, while kaempferol, fisetin, flavone, and myricetin have a weak but consistent association, and other compounds have a minimal association in both embryonic stem (ES) and hematopoietic stem cell (HSC) populations. The frequency of translocations induced by bioflavonoids at later stages of myelopoiesis is significantly reduced by more than one log. The MLL and AF9 bcrs are sensitive to these agents and recombinogenic independent of their native context suggesting bcr sequences themselves are drivers of illegitimate DNA repair reactions and translocations, not generation of functional oncogenic fusions. This system provides for rapid systematic screening of relative risk, dose dependence, and combinatorial impact of multitudes of dietary and environmental exposures on MLL-AF9 translocations. Environ. Mol. Mutagen. 60: 154-167, 2019. © 2018 Wiley Periodicals, Inc.

Downregulation of HOXC6 in Serous Ovarian Cancer.

Homeobox (HOX) genes encode transcription factors critical to morphogenesis and cell differentiation. Although dysregulation of several HOX genes in ovarian cancer has been reported, little is known about HOXC6 expression in epithelial ovarian cancer. In this report, analysis of laser capture microdissected samples determined HOXC6 expression patterns in normal versus malignant serous ovarian carcinoma tissues. HOXC6 protein was quantified by ELISA in parallel serum samples and further validated in a larger cohort of serum samples collected from women with and without serous ovarian carcinoma. These data demonstrate significant downregulation of HOXC6 in serous ovarian cancer.

Aptamer-Functionalized Nanoparticles as "Smart Bombs": The Unrealized Potential for Personalized Medicine and Targeted Cancer Treatment.

Conventional delivery of chemotherapeutic agents leads to multiple systemic side effects and toxicity, limiting the doses that can be used. The development of targeted therapies to selectively deliver anti-cancer agents to tumor cells without damaging neighboring unaffected cells would lead to higher effective local doses and improved response rates. Aptamers are single-stranded oligonucleotides that bind to target molecules with both high affinity and high specificity. The high specificity exhibited by aptamers promotes localization and uptake by specific cell populations, such as tumor cells, and their conjugation to anti-cancer drugs has been explored for targeted therapy. Advancements in the development of polymeric nanoparticles allow anti-cancer drugs to be encapsulated in protective nonreactive shells for controlled drug delivery with reduced toxicity. The conjugation of aptamers to nanoparticle-based therapeutics may further enhance direct targeting and personalized medicine. Here we present how the combinatorial use of aptamer and nanoparticle technologies has the potential to develop "smart bombs" for targeted cancer treatment, highlighting recent pre-clinical studies demonstrating efficacy for the direct targeting to particular tumor cell populations. However, despite these pre-clinical promising results, there has been little progress in moving this technology to the bedside.

Oxidative stress, bone marrow failure, and genome instability in hematopoietic stem cells.

Reactive oxygen species (ROS) can be generated by defective endogenous reduction of oxygen by cellular enzymes or in the mitochondrial respiratory pathway, as well as by exogenous exposure to UV or environmental damaging agents. Regulation of intracellular ROS levels is critical since increases above normal concentrations lead to oxidative stress and DNA damage. A growing body of evidence indicates that the inability to regulate high levels of ROS leading to alteration of cellular homeostasis or defective repair of ROS-induced damage lies at the root of diseases characterized by both neurodegeneration and bone marrow failure as well as cancer. That these diseases may be reflective of the dynamic ability of cells to respond to ROS through developmental stages and aging lies in the similarities between phenotypes at the cellular level. This review summarizes work linking the ability to regulate intracellular ROS to the hematopoietic stem cell phenotype, aging, and disease.

Double-strand break repair by interchromosomal recombination: an in vivo repair mechanism utilized by multiple somatic tissues in mammals.

Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G(2)). However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes-two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit-granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation.

Long-term effects of chromatin remodeling and DNA damage in stem cells induced by environmental and dietary agents.

The presence of histones acts as a barrier to protein access; thus chromatin remodeling must occur for essential processes such as transcription and replication. In conjunction with histone modifications, DNA methylation plays critical roles in gene silencing through chromatin remodeling. Chromatin remodeling is also interconnected with the DNA damage response, maintenance of stem cell properties, and cell differentiation programs. Chromatin modifications have increasingly been shown to produce long-lasting alterations in chromatin structure and transcription. Recent studies have shown environmental exposures in utero have the potential to alter normal developmental signaling networks, physiologic responses, and disease susceptibility later in life during a process known as developmental reprogramming. In this review we discuss the long-term impact of exposure to environmental compounds, the chromatin modifications that they induce, and the differentiation and developmental programs of multiple stem and progenitor cell types altered by exposure. The main focus is to highlight agents present in the human lifestyle that have the potential to promote epigenetic changes that impact developmental programs of specific cell types, may promote tumorigenesis through altering epigenetic marks, and may be transgenerational, for example, those able to be transmitted through multiple cell divisions.