ALBERT

Description: Recently, we and others have shown that protein-protein interactions play an important role in the pathogenesis of leukemia, and that the transcription factor C/EBPα is a key player in granulopoiesis and leukemogenesis. In the present study, we sought to identify C/EBPα interacting proteins. A glutathione-S-transferase-C/EBPα fusion protein was used to pull down interacting proteins from U937 nuclear extracts. These proteins were analyzed by 2-D gel electrophoresis, 1-D nano LC and identified by MALDI or MALDI-TOF/TOF. Several novel C/EBPα interactors were identified including known proteins like Rb, hnRNP and E2F4. Two novel interactions, one of cell cycle regulator protein MCM5 and the other with the MYST domain histone acetyl transferase TIP60 with C/EBPα were further confirmed by using pull-down and co-immunoprecipitation experiments. TP60 was able to markedly enhance C/EBPα mediated transcription in reporter gene assays, suggesting that TIP60 is a co-activator of C/EBPα. This co-activator function of TIP60 was dependent on an intact histone acetyl transferase domain and on the C/EBPα DNA binding domain. TIP60 was found to be associated with the human C/EBPα promoter in-vivo in a chromatin immunoprecipitation assay with a concomitant increase in histone H3 and H4 acetylation. Furthermore, we observed a lower expression of TIP60 mRNA in U937 CD11b− compared to retinoic acid induced U937 CD11b+ cells suggesting that higher TIP60 expression is associated with myeloid differentiation. There was also a marked correlation between the expression levels of TIP60 and C/EBPa in normal bone marrow, chronic myeloid leukemia and acute myeloid leukemia samples with t(8;21), inv(16) and t(15;17). This finding further confirms the functional synergism between C/EBPα and TIP60 and suggests that TIP60 might be an important player in leukemogenesis.

Description: In this report, we show that the Src family nonreceptor protein tyrosine kinase (PTK) Lyn associates with aggregated IgA Fc receptor (FcαR) in the monocytic cell line THP-1. Receptor aggregation and subsequent immunoprecipitation of receptor complexes with huIgA adsorbed to nitrocellulose particles shows that Lyn associates with FcαR by a mechanism sensitive to short treatment with the Src family-selective inhibitor PP1. However, interaction of Lyn with IgG Fc receptor (FcγR) in THP-1 cells was unaffected by short treatment with the PTK inhibitor. Cross-linking of FcαR induced tyrosine phosphorylation of several cellular proteins, including p72Syk, which appears to be a major target of early PTK activity. Unexpectedly, in vitro kinase assays showed that FcαR aggregation-induced tyrosine phosphorylation of Syk did not result in upregulation of Syk activity. Despite the lack of enhanced Syk kinase activity, downstream signaling after FcαR cross-linking was functional and induced the release of significant amounts of interleukin-1 receptor antagonist and interleukin-8. The induction of cytokine release was completely blocked by PP1, thus confirming the biological significance of the association of Lyn with aggregated FcαR. Our data show that early signal transduction after FcαR cross-linking as well as FcαR-mediated activation of cellular effector functions depends on Src family kinase activity. The Src-family PTK involved in FcαR-mediated tyrosine phosphorylation appears to be Lyn, which coprecipitated with aggregated FcαR complexes.

Description: Background: Wiskott-Aldrich syndrome (WAS) is a rare, X-linked, life-threatening primary immunodeficiency caused by mutations in the gene encoding the WAS protein (WASP). WASP-deficient immune cells have compromised immunological synapse formation, cell migration and cytotoxicity. Thus, WAS is characterized by development of recurrent or severe infections, eczema, and increased risk of autoimmunity and malignancies. In addition, WASP deficiency results in microthrombocytopenia, leading to severe bleeding episodes. When a suitable donor is available, WAS can be treated by hematopoietic stem cell transplant (HSCT), but HSCT can be impeded by complications such as graft versus host disease, rejection and autoimmunity. Importantly, HSCT may carry higher risks in older children (〉2-5 yrs) [Shin et al, 2012; Moratto et al, 2011]. An alternative approach is gene therapy (GT). We previously reported interim results of a Phase I/II clinical trial (NCT01515462) in 8 subjects treated with OTL-103, a drug product composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a lentiviral vector (LV) encoding human WASP cDNA under the control of the endogenous promoter [Ferrua et al, 2019]. We now report updated results on the safety and efficacy of OTL-103 in 17 subjects treated at San Raffaele Hospital as part of the same clinical trial or expanded access programs (EAP) with up to 8 yrs follow up (FU). Methods: NCT01515462: As described in Ferrua et al, 8 male subjects (mean age at GT: 4.8 yrs, range 1.1-12.4) were treated with OTL-103. The source of autologous CD34+ HSPCs was bone marrow (BM; n=5), mobilized peripheral blood (mPB; n=2) or both (n=1). As part of a reduced-intensity conditioning regimen, rituximab was given 22 days prior and busulfan + fludarabine during the week before OTL-103 infusion. At time of reporting, all subjects had ≥3 yrs FU (range: 3-8 yrs). EAP: 9 male subjects (11.2 yrs, 1.4-35.1) received identical treatment to subjects in the clinical trial; autologous CD34+ HSPCs source was mPB in all subjects. At time of reporting, subjects had a median of 1.4 yrs FU (range: 0.1-3.0 yrs) with 6/9 having ≥1 yr FU. Results: At last FU for all subjects (median: 3.0 yrs, range 0.1-8.0), overall survival was 94% (16/17). One EAP subject died 4.5 mo post-GT, due to deterioration of an underlying neurodegenerative condition considered unrelated to OTL-103 by investigator. To date, there have been no reports of insertional oncogenesis or replication-competent LV. While most subjects experienced adverse events (AEs) due to the reduced-intensity conditioning regimen (mainly mild or moderate), there were no reports of AEs related to OTL-103. Efficacy endpoints analyses were performed on surviving patients with ≥1 yr FU. Evidence of engraftment of genetically corrected HSPCs and LV+ colonies in BM was observed within 3 mo and persisted up to 8 yrs - the longest published FU of LV vector durability to date (Figure). WASP expression was restored after GT, shown by increases in the fraction of WASP+ lymphocytes and platelets (PLT) within 3 mo and maintained thereafter (Table). After GT, PLT counts improved, leading to a reduction of frequency and severity of bleeding events. Independence from PLT transfusions and absence of severe bleeding events were observed in all subjects by 9 mo FU (Table). Immune function improved; all evaluable patients discontinued immunoglobulin supplementation after GT (median time to discontinuation: 0.9 years after GT, range: 0.2-5 years). Furthermore, reduction in severe infection rate was observed post-GT, suggestive of immune reconstitution (Table). The decrease in bleeding events and severe infection rates occurred despite the integration of subjects into normal daily activities. Eczema progressively resolved or was reduced compared to baseline. Conclusions: This combined analysis of 17 subjects treated in a clinical trial or EAP with up to 8 yrs FU demonstrates that GT continues to be an effective treatment for WAS. All surviving subjects achieved high levels of multilineage engraftment, sustained restoration of WASP expression in lymphocytes and PLTs, improved PLT counts, and fewer bleeding events. A significant reduction in severe infection rate suggests reconstitution of immune function. Importantly, clinical benefit was also attained in older subjects (〉5 yrs), a group considered at higher risk when treated with allogeneic HSCT. Disclosures Jones: Orchard Therapeutics: Employment, Equity Ownership. Dott:Orchard Therapeutics: Employment, Equity Ownership. Naldini:Genenta Science: Consultancy, Equity Ownership; Magenta Therapeutics: Equity Ownership; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was then licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.. Aiuti:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was than licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Study PI.

Description: The 26 December 2004 tsunami severely affected Somalia, with some 300 deaths at a distance of 5,000 km from the epicenter of the magnitude 9.0 earthquake. Somalia's physical characteristics allowed a detailed assessment of the far-field impact of a tsunami in the main propagation direction. The UNESCO mission surveyed five impacted towns south of the Horn of Africa along the Puntland coast in northern Somalia: Eyl, Bandarbeyla, Foar, Xaafuun, and Bargaal. The international team members visited Somalia during 2–10 March 2005. The team measured tsunami runup heights and local flow depths on the basis of the location of watermarks on buildings and eyewitness accounts. Maximum runup heights were typically on the order of 5–9 m. Each measurement was located by means of global positioning systems (GPS) and was photographed. Numerous eyewitness interviews were recorded on video.

Description: The effects of the 26 December 2004 Indian Ocean tsunami on the island of Madagascar were surveyed in July and August of 2005. Runup and inundation were obtained at 52 sites, covering most of the eastern coast of the country, ranging from a maximum runup of 5.4 m in the south to locations where the tsunami was not observed by eyewitnesses present on the day of the event. The data set is characterized by significant heterogeneity, suggesting the importance of local factors in controlling runup. The report of a 50-m vessel breaking its moorings in the port of Toamasina several hours after the maximum visible activity of the wave underscores the complexity of harbor responses and the need to re-evaluate civil defense policies in port environments. Important factors are how the Malagasy population responded to the warning issued during the Nias earthquake, on 28 March 2005, and the hazard posed to Madagascar by possible future mega-earthquakes in south Sumatra.

Description: The tsunami of 26 December 2004 severely affected the Maldives at a distance of 2,500 km from the epicenter of the magnitude 9.0 earthquake. The Maldives provide an opportunity to assess the impact of a tsunami on coral atolls. Two international tsunami survey teams (ITSTs) surveyed a total of 13 heavily damaged islands. The islands were visited by seaplane on 14–15 and 18–19 January 2005. We recorded tsunami heights of up to 4 m on Vilufushi on the basis of the location of debris in trees and watermarks on buildings. Each watermark was localized by means of a global positioning system (GPS) and was photographed. Numerous eyewitness interviews were recorded on video. The significantly lower tsunami impact on the Maldives as compared with Sri Lanka is largely due to the topography and bathymetry of the atoll chain.

Description: In August 2005, a team surveyed the effects of the December 2004 Indian Ocean tsunami on the southern coast of Oman. Runup and inundation were obtained at 41 sites, extending over a total of 750 km of shoreline. Measured runup ranged from 3.25 m in the vicinity of Salalah to a negligible value at one location on Masirah Island. In general, the largest values were found in the western part of the surveyed area. Significant incidents were documented in the port of Salalah, where a 285-m-long vessel broke its moorings and drifted inside and outside the port, and another ship struck the breakwater while attempting to enter the harbor. The general hazard to Oman from tsunamis may be greatest from the neighboring Makran subduction zone in western Pakistan.

Description: Acute Myeloid Leukemia (AML) is characterized by specific cytogenetic aberrations which are strong determinants of prognostic outcome and therapeutic response. Because the clinical outcome in AML cytogenetic groups differs considerably, we hypothesized that cytogenetic risk groups of AML might differ specifically in their proteome, protein interaction pathways and posttranslational modifications (PTMs). Thus, we determined the proteome of 30 AML patients belonging to various cytogenetic groups based on two-dimensional gel electrophoresis and Nano LC coupled MALDI-TOF-TOF tandem mass spectrometry. We could identify substantial differences in the proteome, protein expression and peak pattern between cytogenetic risk groups of AML. The interactome analysis based on computational bioinformatics using Ingenuity analysis revealed major regulating networks: MAPK8 and MYC for complex aberrant karyotype AML, TP53 for t(8;21)-AML, TP53- MYC- PRKAC for 11q23-AML, JUN and MYC for inv(16)-AML. Most interestingly, peak explorer analysis revealed a modification of O-linked acetyl glucosamine of hnRNPH1 in AML patients with a 11q23 translocation, an acetylation of calreticulin in t(8;21) translocation AML, an increased intensity of dimethylated peptide of hnRNPA2/B1 in AML patients with translocations of t(8;21) and inv(16) in comparison to healthy bone marrow. We show for the first time that cytogenetic risk groups of AML differ specifically both in their proteome, interactome and PTMs. These findings lead to a new thinking about the pathogenesis of AML and has major therapeutic implications because PTMs are the primary drug targets.

Description: In this report, we show that the Src family nonreceptor protein tyrosine kinase (PTK) Lyn associates with aggregated IgA Fc receptor (FcαR) in the monocytic cell line THP-1. Receptor aggregation and subsequent immunoprecipitation of receptor complexes with huIgA adsorbed to nitrocellulose particles shows that Lyn associates with FcαR by a mechanism sensitive to short treatment with the Src family-selective inhibitor PP1. However, interaction of Lyn with IgG Fc receptor (FcγR) in THP-1 cells was unaffected by short treatment with the PTK inhibitor. Cross-linking of FcαR induced tyrosine phosphorylation of several cellular proteins, including p72Syk, which appears to be a major target of early PTK activity. Unexpectedly, in vitro kinase assays showed that FcαR aggregation-induced tyrosine phosphorylation of Syk did not result in upregulation of Syk activity. Despite the lack of enhanced Syk kinase activity, downstream signaling after FcαR cross-linking was functional and induced the release of significant amounts of interleukin-1 receptor antagonist and interleukin-8. The induction of cytokine release was completely blocked by PP1, thus confirming the biological significance of the association of Lyn with aggregated FcαR. Our data show that early signal transduction after FcαR cross-linking as well as FcαR-mediated activation of cellular effector functions depends on Src family kinase activity. The Src-family PTK involved in FcαR-mediated tyrosine phosphorylation appears to be Lyn, which coprecipitated with aggregated FcαR complexes.