A lentiviral system for RNAi transgenesis and the Ena/VASP triple-knockout defines neuronal and non-neuronal functions in mouse development

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005.Vita.Includes bibliographical references.Mammalian development extends and exploits signaling pathways that function exclusively in axon guidance in lower organisms. This emerging paradigm employs complex expression patterns of expanded protein families to achieve the complexity and specificity required in mammalian development. For example, the Drosophila axon guidance ligands, Netrin and Slit, have recently been implicated in the development of several mammalian organ systems. While the characterization of extra-neuronal functions of ligands and receptors has emerged, the conservation of intracellular signaling pathways remains unclear. The Ena/VASP protein family is a common downstream effector of multiple axon guidance signaling cascades. The analysis of the Ena/VASP triple-null mouse allows us to determine the extent to which these intracellular cascades have been conserved in the development of the mammalian nervous system as well as other organs. Within the nervous system, we have uncovered novel roles for Ena/VASP in the initiation of axon extension, guidance of non-commissural axons, and neuronal migration. Outside the nervous system, we have observed a novel role for Ena/VASP in blood vessel physiology.(cont.) Interestingly, several developmental pathways for which axon guidance receptors have been implicated appear to develop normally in Ena/VASP triple-null embryos. Future work in Ena/VASP developmental biology will analyze the specific roles of Ena/VASP splice isoforms and unique functions of individual Ena/VASP family members. I have developed a lentiviral system for the creation of mouse transgenics including RNAi knockdowns that can be applied to address these questions.by Douglas A. Rubinson.Ph.D

ARF Is Not Required for Apoptosis in Rb Mutant Mouse Embryos

AbstractThe retinoblastoma (RB) tumor suppressor gene occupies central roles in cell cycle control and tumor suppression [1]. Homozygous mutant (Rb−/−) embryos die at E13.5–E15.5 [2–4], exhibiting extensive apoptosis and inappropriate S phase entry in the central and peripheral nervous systems, liver, and ocular lens [2–6]. Mice simultaneously mutant for Rb and other genes can be generated to assess the requirement for these genes in cell cycle control and apoptosis. Using such analysis, E2f-1, E2f-3, p53, and Id2 have been identified as important regulators of cell cycle control and apoptosis in Rb−/− embryos [7–10]. Because unrestrained E2F activity in the absence of Rb function contributes to p53-dependent apoptosis in many systems [7, 9, 11–14], we wished to identify genes linking deregulated E2F activity to p53 activation and subsequent apoptosis. As a transcriptional target of E2F-1 [15–18], a regulator of p53 [19–21], and an important mediator of apoptosis [20–26], ARF was a strong candidate for such a role, especially since it can be upregulated in the absence of Rb[21]. From the analysis of Rb/ARF compound mutants we demonstrate that ARF is not an obligatory link between Rb inactivation and p53-dependent apoptosis

Ena/VASP is required for endothelial barrier function in vivo

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are key actin regulators that localize at regions of dynamic actin remodeling, including cellular protrusions and cell–cell and cell–matrix junctions. Several studies have suggested that Ena/VASP proteins are involved in the formation and function of cellular junctions. Here, we establish the importance of Ena/VASP in endothelial junctions in vivo by analysis of Ena/VASP-deficient animals. In the absence of Ena/VASP, the vasculature exhibits patterning defects and lacks structural integrity, leading to edema, hemorrhaging, and late stage embryonic lethality. In endothelial cells, we find that Ena/VASP activity is required for normal F-actin content, actomyosin contractility, and proper response to shear stress. These findings demonstrate that Ena/VASP is critical for actin cytoskeleton remodeling events involved in the maintenance of functional endothelia

A Lentivirus-Mediated Genetic Screen Identifies Dihydrofolate Reductase (DHFR) as a Modulator of β-Catenin/GSK3 Signaling

The multi-protein β-catenin destruction complex tightly regulates β-catenin protein levels by shuttling β-catenin to the proteasome. Glycogen synthase kinase 3β (GSK3β), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark β-catenin for ubiquitination and subsequent degradation. Because modulation of both β-catenin and GSK3β activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the β-catenin/GSK3β interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a β-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3′oxime (BIO), a specific inhibitor of GSK3β. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of β-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear β-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3β and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFα, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of β-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease

U snRNP assembly in yeast involves the La protein

In all eukaryotic nuclei, the La autoantigen binds nascent RNA polymerase III transcripts, stabilizing these RNAs against exonucleases. Here we report that the La protein also functions in the assembly of certain RNA polymerase II-transcribed RNAs into RNPs. A mutation in a core protein of the spliceosomal snRNPs, Smd1p, causes yeast cells to require the La protein Lhp1p for growth at low temperatures. Precursors to U1, U2, U4 and U5 RNAs are bound by Lhp1p in both wild-type and mutant cells. At the permissive temperature, smd1–1 cells contain higher levels of stable U1 and U5 snRNPs when Lhp1p is present. At low temperatures, Lhp1p becomes essential for the accumulation of U4/U6 snRNPs and for cell viability. When U4 RNA is added to extracts, the pre-U4 RNA, but not the mature RNA, is bound by Smd1p. These results suggest that, by stabilizing a 3′-extended form of U4 RNA, Lhp1p facilitates efficient Sm protein binding, thus assisting formation of the U4/U6 snRNP

Pancreatic Adenocarcinoma: Real World Evidence of Care Delivery in AccessHope Data

Background: Pancreatic adenocarcinoma is an aggressive disease and the delivery of comprehensive care to individuals with this cancer is critical to achieve appropriate outcomes. The identification of gaps in care delivery facilitates the design of interventions to optimize care delivery and improve outcomes in this population. Methods: AccessHope™ is a growing organization that connects oncology subspecialists with treating providers through contracts with self-insured employers. Data from 94 pancreatic adenocarcinoma cases (August 2019–December 2022) in the AccessHope dataset were used to describe gaps in care delivery. Results: In all but 6% of cases, the subspecialist provided guideline-concordant recommendations anticipated to improve outcomes. Gaps in care were more pronounced in patients with non-metastatic pancreatic cancer. There was a significant deficiency in germline testing regardless of the stage, with only 59% of cases having completed testing. Only 20% of cases were receiving palliative care or other allied support services. There was no difference in observed care gaps between patients receiving care in the community setting vs. those receiving care in the academic setting. Conclusions: There are significant gaps in the care delivered to patients with pancreatic adenocarcinoma. A concurrent subspecialist review has the opportunity to identify and address these gaps in a timely manner

Emerging Role of Targeted Therapy in Metastatic Pancreatic Adenocarcinoma

The aggressive biology of pancreatic ductal adenocarcinoma (PDAC), along with its limited sensitivity to many systemic therapies, presents a major challenge in the management of patients with metastatic PDAC. Over the past decade, the incorporation of combinatorial cytotoxic chemotherapy regimens has improved patient outcomes. Despite these advances, resistance to cytotoxic chemotherapy inevitably occurs, and there is a great need for effective therapies. A major focus of research has been to identify molecularly defined subpopulations of patients with PDAC who may benefit from targeted therapies that are matched to their molecular profile. Recent successes include the demonstration of the efficacy of maintenance PARP inhibition in PDAC tumors harboring deleterious BRCA1, BRCA2, and PALB2 alterations. In addition, while therapeutic targeting of KRAS was long thought to be infeasible, emerging data on the efficacy of KRAS G12C inhibitors have increased optimism about next-generation KRAS-directed therapies in PDAC. Meanwhile, KRAS wild-type PDAC encompasses a unique molecular subpopulation of PDAC that is enriched for targetable genetic alterations, such as oncogenic BRAF alterations, mismatch repair deficiency, and FGFR2, ALK, NTRK, ROS1, NRG1, and RET rearrangements. As more molecularly targeted therapies are developed, precision medicine has the potential to revolutionize the treatment of patients with metastatic PDAC