ACKNOWLEDGEMENTSFirst off, I would like to thank Dr. Donald Miller for taking me on as a co-op student and introducing me to the vitally important and fascinating world of cancer research. He not only helped me discover my dream job, but he has also set an outstanding example of how to be an extraordinary physician scientist that I one day hope to take after. I also want to thank all of the members of the Miller/Bates lab who have helped me learn how fun life can be as a scientist and have inspired me to keep improving no matter what. Thank you to Sheila Thomas for her help with the stability experiments, and to Dr. Tariq Malik for his help with the in vivo imaging studies. Special thanks to Dr. Kara Sedoris, who had the patience and fortitude to work with me every day. I enjoyed every minute of it, and couldn't have done it without her! I also want to thank Dr. Frieboes and Dr. Panchapakesan for being part of my committee. I appreciate all of your hard work and advice which has been extremely helpful. I also want to thank Dr. Andre Gobin for being such a great professor and for sponsoring my independent study, which was a very enjoyable experience! To determine the biological role of VEGFq on non-small cell lung cancer in vivo, A549 cells were injected into nude mice and grown for 10 days prior to daily IP injections of 10 mg/kg VEGFq or the control vehicle for 14 days. Tumor progression was physically measured using calipers three times a week. Fluorescent imaging was used to detect the presence, stability, and distribution of Alexa Fluor 750-labeled VEGFq after injection into the mouse to ensure localization in the targeted tumor.These results demonstrate that A549 cells treated with VEGF quadruplex-forming oligonucleotides experience a dramatic decrease in cell proliferation, suggesting that VEGFq may have significant therapeutic implications for the treatment of non-small cell lung cancer.
Mammalian erythropoiesis has long been established to occur within erythroblastic islands (EBIs), niches where erythroblasts differentiate in close contact with a central macrophage. While it is generally accepted that EBI macrophages play an important role in regulation of erythropoiesis, very little is known about the specific macrophage populations involved in EBI formation, the regulation that occurs within EBIs, or how this niche fits into the broader context of hematopoiesis. We analyzed native EBIs isolated from mouse bone marrow using multispectral imaging flow cytometry (Seu et. al. Front Immunol 2017). Consistent with historical observations, the EBIs were heterogeneous and many contained a number of closely CD11b+ cells in addition to erythroblasts and a central F4/80+ macrophage. Flow cytometry analysis of cells dissociated from native bone marrow EBIs indicated these niches are also enriched 2-3 fold in myeloblasts and granulocytic precursors up to metamyelocytes relative to the total bone marrow while they are depleted of mature granulocytes (bands and segmented cells). Bulk RNAseq of the CD11b+ population isolated from EBIs showed high expression of genes characteristic of the granulocytic lineage (e.g. Elane, Mpo, Gfi1, Cebpe, Camp, and Mmp9), indicating the EBI macrophages may regulate myelopoiesis along with erythropoiesis and that EBIs should really be considered as erythro-myeloblastic islands (EMBIs). To critically document the various hematopoietic cell populations that constitute EMBIs, we used the 10x Genomics Chromium system to obtain single cell gene expression data on ~3,500 total cells from isolated EMBIs along with at least 1,000 sorted cells from each of the 3 major EMBI-associated populations (F4/80+, CD71+, and CD11b+) (Fig 1a, b). The data were analyzed using 10x Genomics' Loupe cell Browser and Iterative Clustering and Guide-gene Selection (ICGS, http://www.AltAnalyze.org, Olsson et. al. Nature 2016). From the ICGS analysis, ~30% of the total EMBI-associated cells were myeloid cells that segregated into at least 3 transcriptionally distinct clusters representing granulocytic progenitors and precursors. As expected, erythroblasts with a progressive maturation pattern made up the bulk (60%) of the EMBI-associated cells, while up to 10% were a heterogeneous population of cells that exhibited expression of macrophage markers such as Csf1R and Irf8, along with genes previously described to characterize resident macrophages, such as Fn1and Fsp1/S100A4 (Fig 1c). In order to investigate the balance of myeloid cells with erythroid cells within the EMBIs, we examined the ratio of CD71+ cells to CD11b+ and how this ratio changes in models of altered granulopoiesis. While the number of myeloid cells at any island varied, the overall ratio of CD11b+ area to CD71+ within the EMBIs was relatively constant at steady state. In three different murine models of anemia of inflammation (AoI), we found that this ratio of CD11b+ to CD71+ cells within the EMBI increases dramatically indicating that the increased granulopoiesis and suppression of erythropoiesis noted in AoI is a result of altered balance of the hematopoiesis within the EMBI unit. Similarly, stimulation of granulopoiesis with GCSF also results in a shift within the EMBIs to CD11b+ myeloid cells and suppression of erythroid cells. Alternatively, in gfi1 KO mice, a model of congenital neutropenia in which granulopoiesis fails at an early stage, the ratio shifts toward CD71+ erythroid cells with paucity of the granulocytic precursors that are typically found at the EMBIs. Taken together, these data indicate that granulocyte progenitors and precursors are specifically associated with EMBI macrophages in the mouse bone marrow. The preferential localization of myeloid precursors within EMBIs suggests this niche is a site for granulopoiesis as well as erythropoiesis and production of these lineages is dynamically regulated within this niche. Our work with multiple murine models of altered granulopoiesis demonstrates that pathological expansion of one of the lineages within this niche may suppress the other and that the interactions within the EMBI could be a useful therapeutic target for AoI. These novel findings significantly broaden our understanding of the role of this hematopoietic niche in the regulated development of lineage committed erythroid and myeloid cells. Disclosures No relevant conflicts of interest to declare.
Activating mutations of Rho-family of small GTPases have been linked to lymphoproliferative disorders, although the pathogenesis mechanism involved is unknown. BCR-ABL (p190) B-cell acute lymphoblastic leukemia (B-ALL) arises from the expression of the oncofusion protein BCR-ABL in a B-cell progenitor. The transforming effect of BCR-ABL is dependent on the tyrosine kinase (TK) activity of the fusion protein that leads to autophosphorylation, recruitment of adaptor proteins, and subsequent activation of downstream signaling. TK inhibitors (TKIs) have been used as frontline treatment for Ph+ B-ALL patients. However, relapse is common in Ph+ B-ALL despite high rates of complete response with initial therapy, probably because of survival of leukemic progenitors. These BCR-ABL+ progenitors appear to develop additional epigenetic and genetic alterations that result in proliferative advantage frequently associated with silencing of the cyclin dependent kinase inhibitor Cdkn2a, even before mutant Cdkn2a gene deleted cells are selected during clonal evolution. Recent work by our group (Chang KH et al., Blood 2012) identified the Rho GTPase guanine nucleotide exchange factor Vav3 in BCR-ABL mediated lymphoid leukemogenesis. We showed that the deficiency of the guanosine nucleotide exchange factor Vav3 delays leukemogenesis and phenocopies the effect of Rac2 (and combined Rac2/Rac1) deficiency (Thomas EK et al., Cancer Cell 2007; Sengupta A et al., Blood 2010), a downstream effector of Vav3. Upregulated Vav3 expression and activation only partly depend on ABL TK activity, and Vav3 deficiency collaborates with tyrosine kinase inhibitors to impair leukemogenesis in vitro and in vivo through impaired proliferation and survival. On the other hand, our group has demonstrated that Bmi1 overexpression frequently found in BCR-ABL+ B-ALL results in B-cell progenitor reprogramming through acquisition of a stem cell-like phenotype (Sengupta A et al., Blood 2012). Bmi1 forms part of the classical polycomb repression complex 1 (PRC1) where its component Ring1A/B catalyzes histone H2A mono-ubiquitination at lysine 119, which in conjunction with the PRC2 complex activity leads to chromatin compaction and repression of target genes. Through epistasis experiments, we found that Vav3 or Rac2 deficiency abrogates the oncogenic effect of Bmi1 overexpression. Co-immunoprecipitation experiments in nuclear and cytoplasmic cell extracts demonstrated that Vav3 and Rac1/Rac2 co-immunoprecipitate with Bmi1 in the nucleus but not in the cytosol of BCR-ABL+ leukemic cells. Interestingly, control non-BCR-ABL expressing nuclear extracts show minimal, if any, level of co-immunoprecipitation. This co-immunoprecipitation is not directly induced by BCR-ABL since BCR-ABL does not co-immunoprecipitate with Vav3/Rac1/Rac2 but does with Bmi1, suggesting that nuclear Vav3 activity may be dissected from the TK activity of BCR-ABL. Biochemically, the overexpression of Bmi1 results in increased activation of nuclear Rac which is practically abrogated by the deficiency of Vav3 as assessed in cellular pulldown assays of primary leukemic B-cell progenitors. As expected, downstream expression of Cdkn2a is repressed by overexpression by Bmi1. Deficiency of Vav3 restores the expression of Cdkn2a to control levels. This data suggests a transcriptional regulatory role of the signaling proteins Vav3/Rac2 in the nucleus. Chromatin immunoprecipitation (ChIP)-qPCR for Bmi1, Ring1B and polycomb repressive histone marks (H2AK119 and H3K27me3) and the assay for Tn5-transposase accessible chromatin (ATAQ)-qPCR for the Cdkn2a locus in Vav3- or Rac2-deficient, BCR-ABL+ primary B-cell progenitors were compared with their BCR-ABL, Vav3/Rac2 expressing counterparts. These assays confirmed that Vav3 and Rac2 are essential for PRC dependent transcriptional repression of Cdkn2a through occupancy of the Cdkn2a promoter and decreased accessibility to Cdkn2a chromatin. In conclusion, our studies establish for the first time an association between nuclear Vav3/Rac and polycomb repressive activities in p190-BCR-ABL+ leukemogenesis through their activity on the Cdkn2a locus. Vav3 may represent a novel target for adjuvant therapy with TKI in BCR-ABL+ lymphoblastic leukemia. Disclosures No relevant conflicts of interest to declare.
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