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Open Access Publications from the University of California

Scholarly Works (11 results)

  • Article
  • Peer Reviewed
UC San Francisco Previously Published Works (2015)
Time-lapse imaging can be used to quantify how cells move, divide, and die over time and under defined culture conditions. Open source software packages such as CellProfiler, Icy, and Fiji provide robust and convenient interfaces for performing such analyses. However, object tracking algorithms are imperfect, and validation of significant events is often required. This is challenging, as CellProfiler produces only tabular data for object tracking, and the graphical tools in Icy and Fiji are not optimal for manual review of these events. Here we describe Traxtile, a program that allows interactive graphical review and revision of object tracking assignments. Traxtile imports initial assignments and automatically identifies events needing review (i.e., apparent creation of new objects, splits, merges, and losses). For each such event, the object track is displayed on a montage of images centered on the event and spanning the preceding and subsequent frames. Links between cells in successive frames can be reviewed and edited, yielding validated tracks for the image series. Reports summarize events from the validated tracks. Traxtile is implemented in Python version 2.7 using standard distribution libraries (available at www.python.org) and is freely available at https://github.com/braunb/traxtile-public.
    Cover page: Traxtile: Interactive editing of cell tracks in time-lapse images
    • Thesis
    • Peer Reviewed
    UC San Francisco Electronic Theses and Dissertations (2012)

    Juvenile myelomonocytic leukemia (JMML) is a rare childhood myeloproliferative neoplasm/myelodysplastic syndrome associated with mutations in the Ras/mitogen activated protein kinase (MAPK) signaling pathway. PTPN11 is the most commonly mutated gene in JMML, accounting for 35% of cases, followed by gain-of-function mutations in KRAS and NRAS and loss-of-function mutations in NF1. The protein tyrosine phosphatase (PTP) SHP-2 encoded by the gene PTPN11 is known to play a critical role in hematopoiesis and development, and studies in Drosophila and C. elegans have identified the SHP-2 homologs corkscrew and ptp-2 as crucial mediators of Ras and growth factor receptor signaling. SHP-2 is an SH2-domain containing non-receptor PTP whose activity is basally auto-inhibited by the occlusion of its catalytic cleft by its N-SH2 domain. The most frequently identified PTPN11 mutation results in an E76K substitution that is predicted to interfere with the N-SH2/PTP domain interface, thereby disrupting the auto-inhibited conformation. SHP-2 is unusual for a PTP in that it positively regulates growth factor signaling, and the mutations identified in JMML are gain-of-function mutations resulting in a basal increase in catalytic activity. A hallmark feature of JMML is hypersensitivity of hematopoietic progenitors to granulocyte-macrophage colony-stimulating factor (GM-CSF) in an in vitro colony-forming assay (CFU-GM). It has been demonstrated that the catalytic activity of SHP-2 is required for its transforming ability, as substitution of critical catalytic residues abolishes E76K-mediated CFU-GM hypersensitivity. Utilizing PTP substrate-trapping technology, we have used tandem affinity purification and mass spectrometry to identify several putative substrates of SHP-2 E76K, including the protein parafibromin. Examination of changes in global phosphotyrosine mediated by SHP-2 E76K revealed a role for Src family kinases (SFK) in SHP-2 signaling downstream of the GM-CSF receptor, although CFU-GM transformation assays using hematopoietic progenitors from SFK-null murine models suggested that this relationship was not essential for leukemogenesis. Instead, high overexpression of SHP-2 in the human TF-1 leukemia cell line demonstrated a potential role for SHP-2 in promoting megakaryocytic differentiation via inhibition of the SFK Lyn. Lastly, the identification of mutations in C-CBL in a number of hematologic malignancies led us to discover and characterize the role of similar mutations in JMML.

      Cover page: Biochemical Dissection of SHP-2 and C-CBL in Juvenile Myelomonocytic Leukemia
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2021)
      Juvenile myelomonocytic leukemia (JMML) is initiated in early childhood by somatic mutations that activate Ras signaling. Although some patients have only a single identifiable oncogenic mutation, others have 1 or more additional alterations. Such secondary mutations, as a group, are associated with an increased risk of relapse after hematopoietic stem cell transplantation or transformation to acute myeloid leukemia. These clinical observations suggest a cooperative effect between initiating and secondary mutations. However, the roles of specific genes in the prognosis or clinical presentation of JMML have not been described. In this study, we investigate the impact of secondary SH2B3 mutations in JMML. We find that patients with SH2B3 mutations have adverse outcomes, as well as higher white blood cell counts and hemoglobin F levels in the peripheral blood. We further demonstrate this interaction in genetically engineered mice. Deletion of Sh2b3 cooperates with conditional Nf1 deletion in a dose-dependent fashion. These studies illustrate that haploinsufficiency for Sh2b3 contributes to the severity of myeloproliferative disease and provide an experimental system for testing treatments for a high-risk cohort of JMML patients.
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2013)
        Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile myelomonocytic leukemia (JMML), an aggressive myeloproliferative neoplasm (MPN) that is refractory to conventional chemotherapy. Conditional inactivation of the Nf1 tumor suppressor in hematopoietic cells of mice causes a progressive MPN that accurately models JMML and chronic myelomonocytic leukemia (CMML). We characterized the effects of Nf1 loss on immature hematopoietic populations and investigated treatment with the MEK inhibitor PD0325901 (hereafter called 901). Somatic Nf1 inactivation resulted in a marked expansion of immature and lineage-committed myelo-erythroid progenitors and ineffective erythropoiesis. Treatment with 901 induced a durable drop in leukocyte counts, enhanced erythropoietic function, and markedly reduced spleen sizes in mice with MPN. MEK inhibition also restored a normal pattern of erythroid differentiation and greatly reduced extramedullary hematopoiesis. Remarkably, genetic analysis revealed the persistence of Nf1-deficient hematopoietic cells, indicating that MEK inhibition modulates the proliferation and differentiation of Nf1 mutant cells in vivo rather than eliminating them. These data provide a rationale for performing clinical trials of MEK inhibitors in patients with JMML and CMML.
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2016)
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2024)

            Background

            Neuroblastoma is the most common extra-cranial pediatric solid tumor. 131I-metaiodobenzylguanidine (MIBG) is a targeted radiopharmaceutical highly specific for neuroblastoma tumors, providing potent radiotherapy to widely metastatic disease. Aurora kinase A (AURKA) plays a role in mitosis and stabilization of the MYCN protein in neuroblastoma. We aimed to study the impact of AURKA inhibitors on DNA damage and tumor cell death in combination with 131I-MIBG therapy in a pre-clinical model of high-risk neuroblastoma.

            Results

            Using an in vivo model of high-risk neuroblastoma, we demonstrated a marked combinatorial effect of 131I-MIBG and alisertib on tumor growth. In MYCN amplified cell lines, the combination of radiation and an AURKA A inhibitor increased DNA damage and apoptosis and decreased MYCN protein levels.

            Conclusion

            The combination of AURKA inhibition with 131I-MIBG treatment is active in resistant neuroblastoma models.
              Cover page: Aurora Kinase A inhibition enhances DNA damage and tumor cell death with 131I-MIBG therapy in high-risk neuroblastoma
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2009)
              How oncogenes modulate the self-renewal properties of cancer-initiating cells is incompletely understood. Activating KRAS and NRAS mutations are among the most common oncogenic lesions detected in human cancer, and occur in myeloproliferative disorders (MPDs) and leukemias. We investigated the effects of expressing oncogenic Kras(G12D) from its endogenous locus on the proliferation and tumor-initiating properties of murine hematopoietic stem and progenitor cells. MPD could be initiated by Kras(G12D) expression in a highly restricted population enriched for hematopoietic stem cells (HSCs), but not in common myeloid progenitors. Kras(G12D) HSCs demonstrated a marked in vivo competitive advantage over wild-type cells. Kras(G12D) expression also increased the fraction of proliferating HSCs and reduced the overall size of this compartment. Transplanted Kras(G12D) HSCs efficiently initiated acute T-lineage leukemia/lymphoma, which was associated with secondary Notch1 mutations in thymocytes. We conclude that MPD-initiating activity is restricted to the HSC compartment in Kras(G12D) mice, and that distinct self-renewing populations with cooperating mutations emerge during cancer progression.
                Cover page: Oncogenic Kras initiates leukemia in hematopoietic stem cells.
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2013)
                Oncogenic K-Ras proteins, such as K-Ras(G12D), accumulate in the active, guanosine triphosphate (GTP)-bound conformation and stimulate signaling through effector kinases. The presence of the K-Ras(G12D) oncoprotein at a similar abundance to that of endogenous wild-type K-Ras results in only minimal phosphorylation and activation of the canonical Raf-mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling cascades in primary hematopoietic cells, and these pathways remain dependent on growth factors for efficient activation. We showed that phospholipase C-γ (PLC-γ), PI3K, and their generated second messengers link activated cytokine receptors to Ras and ERK signaling in differentiated bone marrow cells and in a cell population enriched for leukemia stem cells. Cells expressing endogenous oncogenic K-Ras(G12D) remained dependent on the second messenger diacylglycerol for the efficient activation of Ras-ERK signaling. These data raise the unexpected possibility of therapeutically targeting proteins that function upstream of oncogenic Ras in cancer.
                  Cover page: PLC-γ and PI3K Link Cytokines to ERK Activation in Hematopoietic Cells with Normal and Oncogenic Kras
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2017)
                  Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder of childhood caused by mutations in the Ras pathway. Outcomes in JMML vary markedly from spontaneous resolution to rapid relapse after hematopoietic stem cell transplantation. Here, we hypothesized that DNA methylation patterns would help predict disease outcome and therefore performed genome-wide DNA methylation profiling in a cohort of 39 patients. Unsupervised hierarchical clustering identifies three clusters of patients. Importantly, these clusters differ significantly in terms of 4-year event-free survival, with the lowest methylation cluster having the highest rates of survival. These findings were validated in an independent cohort of 40 patients. Notably, all but one of 14 patients experiencing spontaneous resolution cluster together and closer to 22 healthy controls than to other JMML cases. Thus, we show that DNA methylation patterns in JMML are predictive of outcome and can identify the patients most likely to experience spontaneous resolution.
                    Cover page: Genome-wide DNA methylation is predictive of outcome in juvenile myelomonocytic leukemia
                    • Article
                    • Peer Reviewed
                    UC San Francisco Previously Published Works (2020)
                    Somatic KRAS mutations are highly prevalent in many cancers. In addition, a distinct spectrum of germline KRAS mutations causes developmental disorders called RASopathies. The mutant proteins encoded by these germline KRAS mutations are less biochemically and functionally activated than those in cancer. We generated mice harboring conditional KrasLSL-P34Rand KrasLSL-T58I knock-in alleles and characterized the consequences of each mutation in vivo. Embryonic expression of KrasT58I resulted in craniofacial abnormalities reminiscent of those seen in RASopathy disorders, and these mice exhibited hyperplastic growth of multiple organs, modest alterations in cardiac valvulogenesis, myocardial hypertrophy, and myeloproliferation. By contrast, embryonic KrasP34R expression resulted in early perinatal lethality from respiratory failure due to defective lung sacculation, which was associated with aberrant ERK activity in lung epithelial cells. Somatic Mx1-Cre-mediated activation in the hematopoietic compartment showed that KrasP34R and KrasT58I expression had distinct signaling effects, despite causing a similar spectrum of hematologic diseases. These potentially novel strains are robust models for investigating the consequences of expressing endogenous levels of hyperactive K-Ras in different developing and adult tissues, for comparing how oncogenic and germline K-Ras proteins perturb signaling networks and cell fate decisions, and for performing preclinical therapeutic trials.
                      Cover page: KrasP34R and KrasT58I mutations induce distinct RASopathy phenotypes in mice
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