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Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Genes that are aberrantly expressed and/or mutated that lead to the dysplastic erythroid morphology seen in −7/del(7q) MDS have not been identified. In this study, we show that reduced expression of dedicator of cytokinesis 4 ( DOCK4 ) causes dysplasia by disrupting the actin cytoskeleton in developing red blood cells. In addition, our identification of the molecular pathway that leads to morphological defects in this type of MDS provides potential therapeutic targets downstream of DOCK4 that can be exploited to reverse the dysplasia in the erythroid lineage. Furthermore, we developed a novel single-cell multispectral flow cytometry assay for evaluation of disrupted F-actin filaments, which can be used for potential early detection of dysplastic cells in MDS. Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Loss or deletion of chromosome 7 is commonly seen in MDS and leads to a poor prognosis. However, the identity of functionally relevant, dysplasia-causing, genes on 7q remains unclear. Dedicator of cytokinesis 4 (DOCK4) is a GTPase exchange factor, and its gene maps to the commonly deleted 7q region. We demonstrate that DOCK4 is underexpressed in MDS bone marrow samples and that the reduced expression is associated with decreased overall survival in patients. We show that depletion of DOCK4 levels leads to erythroid cells with dysplastic morphology both in vivo and in vitro. We established a novel single-cell assay to quantify disrupted F-actin filament network in erythroblasts and demonstrate that reduced expression of DOCK4 leads to disruption of the actin filaments, resulting in erythroid dysplasia that phenocopies the red blood cell (RBC) defects seen in samples from MDS patients. Reexpression of DOCK4 in −7q MDS patient erythroblasts resulted in significant erythropoietic improvements. Mechanisms underlying F-actin disruption revealed that DOCK4 knockdown reduces ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activation, leading to increased phosphorylation of the actin-stabilizing protein ADDUCIN in MDS samples. These data identify DOCK4 as a putative 7q gene whose reduced expression can lead to erythroid dysplasia.

Transcription factor (TF) DNA-binding dynamics govern cell fate and identity. However, our ability to pharmacologically control TF localization is limited. Here we leverage chemically driven binding site restriction leading to robust and DNA-sequence-specific redistribution of PU.1, a pioneer TF pertinent to many hematopoietic malignancies. Through an innovative technique, ‘CLICK-on-CUT&Tag’, we characterize the hierarchy of de novo PU.1 motifs, predicting occupancy in the PU.1 cistrome under binding site restriction. Temporal and single-molecule studies of binding site restriction uncover the pioneering dynamics of native PU.1 and identify the paradoxical activation of an alternate target gene set driven by PU.1 localization to second-tier binding sites. These transcriptional changes were corroborated by genetic blockade and site-specific reporter assays. Binding site restriction and subsequent PU.1 network rewiring causes primary human leukemia cells to differentiate. In summary, pharmacologically induced TF redistribution can be harnessed to govern TF localization, actuate alternate gene networks and direct cell fate. Chemically driven blockade of PU.1 binding sites leads to its genome-wide redistribution. PU.1 network rewiring causes human acute myeloid leukemia cells to differentiate.

Primary myelofibrosis (PMF) is a clonal hematologic malignancy characterized by BM fibrosis, extramedullary hematopoiesis, circulating CD34+ cells, splenomegaly, and a propensity to evolve to acute myeloid leukemia. Moreover, the spleen and BM of patients harbor atypical, clustered megakaryocytes, which contribute to the disease by secreting profibrotic cytokines. Here, we have revealed that megakaryocytes in PMF show impaired maturation that is associated with reduced GATA1 protein. In investigating the cause of GATA1 downregulation, our gene-expression study revealed the presence of the RPS14-deficient gene signature, which is associated with defective ribosomal protein function and linked to the erythroid lineage in 5q deletion myelodysplastic syndrome. Surprisingly, reduced GATA1 expression and impaired differentiation were limited to megakaryocytes, consistent with a proproliferative effect of a GATA1 deficiency on this lineage. Importantly, expression of GATA1 effectively rescued maturation of PMF megakaryocytes. Together, these results suggest that ribosomal deficiency contributes to impaired megakaryopoiesis in myeloproliferative neoplasms.

Refractory anemia and erythroid dysplasia remain as one of the common clinical presentations and predominant causes of morbidity in patients with the blood malignancy, myelodysplastic syndromes (MDS). Better understanding of mechanisms underlying ineffective erythropoiesis in MDS is critically needed to develop novel therapeutic strategies. Reduced levels of the adaptor protein Dedicator of Cytokinesis 4 (DOCK4) is frequently observed in MDS patients due to epigenetic silencing and/or chromosomal deletions and is associated with dismal prognosis. In this dissertation, I investigated the functional and signaling role of DOCK4 during red blood cell development from a hematopoietic stem cell (HSC). Firstly, my studies have determined that reduced levels of DOCK4 results in erythroid dysplasia. Furthermore, re-expression of DOCK4 in MDS patient samples lacking DOCK4, partially reversed the observed erythroid defects suggesting that re-activation of the DOCK4 pathway might be therapeutically beneficial in MDS patients harboring DOCK4 defects. Secondly, as a means to reactivate the DOCK4 pathway in MDS, I identified targetable signaling networks downstream of DOCK4 by performing unbiased phosphoproteomics using HSCs expressing reduced levels of DOCK4. Finally, I demonstrate avenues for restoring the DOCK4 functions by targeting signaling elements downstream of DOCK4. Specifically, pharmacological inhibition of one of the identified candidates; PTPN6, is capable of relieving the differentiation block along the erythroid lineage in MDS patients expressing reduced levels of DOCK4. In summary, my work has uncovered novel functions and signaling networks regulated by DOCK4 that can be targeted to reverse the aberrant phenotypes arising due to reduced expression of DOCK4 in hematopoietic cells. Most importantly, my work has identified PTPN6 as a potential therapeutic target to alleviate anemia in MDS.

Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Loss or deletion of chromosome 7 is commonly seen in MDS and leads to a poor prognosis. However, the identity of functionally relevant, dysplasia-causing, genes on 7q remains unclear. Dedicator of cytokinesis 4 (DOCK4) is a GTPase exchange factor, and its gene maps to the commonly deleted 7q region. We demonstrate thatDOCK4is underexpressed in MDS bone marrow samples and that the reduced expression is associated with decreased overall survival in patients. We show that depletion ofDOCK4levels leads to erythroid cells with dysplastic morphology both in vivo and in vitro. We established a novel single-cell assay to quantify disrupted F-actin filament network in erythroblasts and demonstrate that reduced expression ofDOCK4leads to disruption of the actin filaments, resulting in erythroid dysplasia that phenocopies the red blood cell (RBC) defects seen in samples from MDS patients. Reexpression of DOCK4 in −7q MDS patient erythroblasts resulted in significant erythropoietic improvements. Mechanisms underlying F-actin disruption revealed thatDOCK4knockdown reduces ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activation, leading to increased phosphorylation of the actin-stabilizing protein ADDUCIN in MDS samples. These data identifyDOCK4as a putative 7q gene whose reduced expression can lead to erythroid dysplasia.