ALBERT

Description: Acute myeloid leukemia (AML) was diagnosed in approximately 18,860 people in the U.S. in 2014, and caused an estimated 10,460 deaths. AML is a heterogeneous clonal disorder with highly varied molecular abnormalities. To permit better prognosis and the development of novel targeted therapies, there is an urgent need to elucidate new molecular targets in AML. Previously, via The Cancer Genome Atlas (TCGA), we had analyzed the molecular and clinical characteristics of 166 AML cases to investigate a prognostic role for miR-199b and demonstrated that low miR-199b levels correlate with worse survival outcomes, especially M5 subtype; however, their correlation with molecular mutations remained unknown. Herein, we attempt to determine the potential cooperative role of NPMc and IDH1 mutations in miR-199b-low AML and investigate the hematological phenotype resulting from miR-199b global knockout mice. Molecular mutations including FLT3-AML, NPMc and IDH1 were analyzed among miR-199b high and low AML cases. Significant correlations in terms of association and survival outcomes were observed for NPMc and IDH1 mutations. Among n=128 AML patients analyzed for miR-199b/NPMc, n=63 were miR-199b-high samples and, of these, n=2 were positive for NPMc mutation. Importantly, of the n=65 miR-199b-low samples, n=41 were positive for NPMc mutation (Fisher p value = 1.941e-08, odds ratio 19.6112). For IDH1 mutation, among the n=139 AML patients analyzed for miR-199b/IDH1, n=59 were miR-199b-high samples (with n=5 positive for IDH1 mutation) whereas n=80 were miR-199b-low samples (with n=25 positive for IDH1 mutation, Fisher p value = 0.01175, odds ratio 3.663005). Combined survival data analysis for miR-199b and NPMc+/IDH1+ (n=169 AML cases) showed worse survival in low miR-199b patients compared to AML patients negative for both mutations with high miR-199b (Chisq = 10.6 on 2 degrees of freedom, p = 0.00491). To define the functional role of miR-199b in myelopoiesis we generated a miR-199b knockout mouse strain via the CRISPR technique. Molecular confirmation of miR-199b silencing, both at the gene and transcript levels,were carried out by PCR-sequencing and RT-qPCR. Peripheral blood analyses at various time points was performed for n=4 mice each; miR-199b knockout mice exhibited a significant increase (p =0.018) in neutrophil levels (1.03 X103/uL) compared to wild type mice (0.72 X103/uL). Eosinophil and lymphocyte levels were also significantly elevated compared to wild type mice (p

Description: Podocalyxin (PodxL) is a CD34 family member previously identified to mark hematopoietic stem cells (HSCs) and other progenitor cells. Previously, we discovered PodxL as a potent erythropoietin (EPO) response gene and demonstrated to promote egression of immature reticulocytes from bone marrow into circulation. PodxL is upregulated in several cancers, including myeloid and lymphoid leukemia. Herein, we aim to define the functional role of PodxL in hematopoiesis - specifically myelopoiesis - by employing conditional PodxL knock out (KO) mouse models. Hematopoietic-specific deletion was achieved using Cre mice with a Vav1 driver and myeloid-specific deletion was achieved with Lyzm2 - Cre driver. We confirmed the deletion of exons 3-7 at the gene, transcript and protein levels using PCR, RT-qPCR and western blotting, respectively. Peripheral blood analysis revealed no difference in blood cell lineages for either KO mouse strain. At steady state, colony forming unit-granulocyte/macrophage (CFU-GM) assay also showed no difference between the KO strains and wild type. In order to examine the functional role of PodxL during stress myelopoiesis, PodxL-/- ; Vav1-Cre mice were treated with 5-Fluorouracil (5FU), a chemotherapeutic agent induces myeloablation. Notably, during rebound of neutrophils, the PodxL-/- ; Vav1-Cre mice showed a sharp increase in neutrophil counts at day 12.5, which at later time points reverted to normal levels comparable to wild type mice. Previously, our in silico analyses combined with outcomes from truncated EpoR knock-in alleles had revealed that PodxL is a potential STAT5 transcriptional target. Here, we tested if G-CSF induces PodxL expression in hematopoietic progenitors. In vivo, G-CSF significantly induced PodxL expression four fold. We then tested the role of PodxL in G-CSF induced neutrophil formation in vivo. Both KO strains (Podxl-/-;Vav1-Cre and Podxl-/-;Lyzm2-Cre) and wild type were treated with G-CSF (125ug/kg/day) for 5 days. Peripheral blood analysis revealed increased neutrophil and monocyte levels in the PodxL-/-;Vav1-Cremice. In order to then determine a possible role of PodxL at the progenitor level, CFU-GM assays were performed. PodxL-/- ; lyzm2-Cre mice had increased colony forming capabilities but there was no difference in PodxL-/-;Vav1-Cre mice compared to wild type. Our results imply that PodxL is playing a negative regulatory role in stress myelopoiesis. Interestingly, the deletion of PodxL in hematopoietic progenitors (Vav1-Cre) resulted in enhanced migration of neutrophils, whereas deletion of PodxL in myeloid compartment (Lyzm2-Cre) resulted in decreased neutrophil migration. This may be in part due to a compensatory effect by CD34 in the hematopoietic compartment. To dissect the molecular mechanism of PodxL during stress myelopoiesis, upon in vivo G-CSF treatment, bone marrow derived hematopoietic progenitors were isolated and PodxL protein was immunoprecipitated. LC-MS/MS proteomic analysis was performed to identify the interacting partners with PodxL. Rap-1A, a small GTPase and member of the RAS family, was among the top interacting proteins. Rap-1A has been shown to promote adhesion and migration of myeloid cells. The association of PodxL with Rap-1A was further confirmed in hematopoietic progenitors by immunoprecipitation and western blotting. To determine if the interaction of PodxL directly regulates Rap-1A activity, a GTP-Rap-1A activity assay was performed in response to G-CSF, GM-CSF and IL-3. Rap-1A activity was significantly elevated in hematopoietic progenitors upon G-CSF treatment in PodxL-/-:Vav1-Cre mice compared to wild type, followed by IL3; however, GM-CSF did not affect Rap-1A activity. In conclusion, our results indicate an important functional role for PodxL in stress myelopoiesis, a function likely mediated via Rap-1A. A complete understanding of the PodxL/Rap-1A axis may reveal important molecular insights into G-CSF-induced mobilization of neutrophils and provide mechanistic understanding into the pathological role of PodxL in aggressive cancers, including leukemia, which in turn may facilitate identification of novel therapeutic targets in PodxL associated cancers. Disclosures No relevant conflicts of interest to declare.

Description: Background: Exosomes are microvesicles that play important roles in intercellular communications in both normal and tumor cells via their cargoes, which include microRNA (miRNA) and proteins. miRNAs play critical regulatory roles in hematopoiesis, and their abnormal expression is correlated with several hematological malignancies, including acute myeloid leukemia (AML). Exosome-derived miRNAs and proteins are increasingly recognized for their prognostic biomarker potential. Exosomes are being evaluated for their potential as novel drug-delivery vehicles due to their endogenous nature and ability to carry small molecule drugs. Aims: We aim to characterize HSC-derived exosomes, investigate the biomarker potential of exosomes derived from AML patient samples (by examining their proteomic and miRNA profiles) and utilize exosomes as innovative drug delivery vehicles with the ability to eliminate leukemic stem cells in a targeted manner. Methods: Secreted exosomes from murine bone marrow HSCs were isolated from conditioned medium and visualized using confocal microscopy. We isolated exosomes and performed miRNA profiling, using qPCR, and LC-MS/MS proteomic analysis to characterize the constituents. We also isolated exosomes from the CD34+ cells of three AML patient samples and profiled 372 of the most abundantly expressed miRs in these cells compared to normal CD34+ cells. We analyzed the proteome of these exosomes as well. In order to assess the utility of exosomes as drug carriers, exosomes from bone marrow-derived OP9 stromal cells were transfected with Daunorubicin (1ug/ul). Normal CD34+ cells and patient-derived AML samples (from n=2 patients) were treated with varying doses of the drug-loaded exosomes. Drug-loaded exosomes uptake was tracked with a Texas Red siRNA. After 24 hours, cells were screened for apoptosis. To test the feasibility of targeted exosomes, OP9 cells were exposed to AML patient samples for 48 hours. The patient cells were then removed and, 24 hours later, "trained" stromal cell-derived exosomes were isolated from the media and transfected with Daunorubicin. These patient-specific, exosomes were plated with both the corresponding patient's CD34+ cells as well as normal CD34+ primary cells. After 24 hours, apoptosis was measured. Results: miRNA profiling of murine bone marrow showed miR-21a, miR-92a and miR-25 were most abundant in exosomes. Proteomic LC-MS/MS analysis revealed presence of exosome-associated novel proteins such as Syntenin-1. Syntenin-1, which is known to bind IL-5R and promote myelopoiesis, was present in significantly higher levels in HSCs compared to myeloid progenitors, implying a functional role for exosome derived Syntenin-1. miRNA profiling in AML samples revealed distinct signature profiles. Importantly, exosome derived miRs such as -1290, -375, -205 and -21-that are known prognostic markers in cancers such as prostate, ovarian and hepatocellular carcinoma-were significantly upregulated in all the three exosome-derived AML samples. The drug-loaded exosomes were successful in inducing significant apoptosis in two patient samples tested. These drug-loaded exosomes also induced cell death in CD34+ normal cells when compared to control exosomes. However, the patient-trained exosomes specifically eliminated 92% of CD34+ AML patient cells, while causing significantly less cell death (44%) of normal CD34+ primary cells exposed to drug-loaded, patient-trained exosomes. Summary/Conclusion: Taken together, our data predict important functional roles for exosome-derived Syntenin-1 in regulating lineage specific hematopoietic differentiations. Furthermore, for the first time, we have identified highly upregulated select exosome-derived miRs from AML patient samples whose prognostic value has been recently reported for other cancers, making these miRs promising candidates for AML biomarkers as well. Finally, stromal cell-derived, drug-loaded exosomes are not only able to induce apoptosis in AML patient samples, but they can effectively be trained by leukemic cells to favor uptake resulting in targeted elimination of leukemic over normal CD34+ cells. Disclosures No relevant conflicts of interest to declare.

Description: Recently, we reported the functional role of miR-199b in AML and its prognostic implications where low miR-199b in AML patients correlated with worse overall survival for the FAB-M5 subtype. Herein, we have attempted to define the role of miR-199b in stress myelopoiesis using knockout mice and examine miR-199b's gene regulation. To define the role of miR-199b and understand its functional role in myeloid proliferation and AML, we generated miR-199b knockout mice via CRISPR approach. The deleted region was confirmed by DNA sequencing and silencing of miR-199b transcripts in nucleated peripheral blood cells was verified via RT-PCR. At steady state, except for significantly elevated neutrophil levels in miR-199b KO mice (n=5) compared to control mice (n=5) at 6 weeks of age, there were no significant differences in peripheral blood profiles. Bone marrow derived HSPCs (hematopoietic stem and progenitor cells) from miR-199b and wild type mice were examined for LT-HSC, KL, LSK, MPP, GMP and MEP populations. At steady state no significant differences were observed in all the cohorts except for KL (Lin-Kit+Sca-) population, where miR-199b KO mice exhibited significantly decreased levels compared to wild type mice (p