The GAB2/SHP2 Pathway in BCR-ABL1-Induced Hematopoietic Neoplasia

BCR-ABL1 is a gene product of the t(9, 22) chromosomal translocation. It encodes a fusion protein with constitutively activated tyrosine kinase activity and is the direct cause of chronic myeloid leukemia (CML) and a subset of B-lymphoid acute lymphoblastic leukemia (B-ALL). ABL1-targeting tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of BCR- ABL1+ hematopoietic neoplasia, but CML patients suffer from persistence of leukemic stem cells (LSCs), and B-ALL patients exhibit poorer response at diagnosis compared with CML patients. Over time, such patients succumb to TKI-resistant leukemic clones. Therefore, additional therapeutic targets are needed to cure these malignancies. I assessed the role of the protein-tyrosine phosphatase (PTP) SHP2 in the initiation and/or maintenance of CML and BCR- ABL1+ B-ALL. I found that SHP2 is required for initiation and maintenance of CML, consistent with its essentiality in normal hematopoietic stem cells (HSCs). In BCR-ABL1+ B-ALL, SHP2 is specifically required for the proliferation of BCR-ABL1+, but not WT, B lymphoid progenitors. The SFK and MEK-ERK pathways downstream of SHP2 independently contribute to this differential requirement. I also assessed the role of GAB2 in downstream signaling from BCR- ABL1, and found that its interaction with SHP2 or PI3K activates different signaling pathways, potentially underlying the differential requirement of these interactions for BCR-ABL1-evoked myeloid and lymphoid malignancies. Furthermore, in a parallel set of studies, I developed a mathematical model to simulate the clinical course of high-grade serous ovarian carcinoma, with potential implications for treatment and screening of this disease. This work is described in the Appendix.Ph.D

Synthesis and Characterization of Lipooligosaccharide-Based Conjugate Vaccines for Serotype B Moraxella catarrhalis

Moraxella catarrhalis is an important cause of otitis media in children and respiratory tract infections in the elderly. Lipooligosaccharide (LOS) is a major surface antigen of the bacterium that elicits bactericidal antibodies. Serological studies show that three major LOS types (A, B, and C) have been identified among clinical isolates. Our previous studies demonstrated that the type A LOS-based conjugates were immunogenic in animals. In this study, LOS from type B strain 26397 was detoxified and conjugated to tetanus toxoid (TT) or a cross-reactive mutant (CRM) of diphtheria toxin to form detoxified LOS (dLOS)-TT and dLOS-CRM, respectively, as vaccine candidates. The molar ratios of dLOS to TT and CRM in the conjugates were 43:1 and 19:1, respectively, while both weight ratios were around 0.9. The antigenicity of the conjugates was similar to that of the LOS, as determined by enzyme-linked immunosorbent assay using a rabbit antiserum to strain 26397. Subcutaneous immunization with each conjugate elicited a 180- to 230-fold rise of serum anti-LOS immunoglobulin G in mice and >2,000-fold rise in rabbits. In addition, both mouse and rabbit antisera showed elevated complement-mediated bactericidal activity against the homologous strain, and a representative rabbit antiserum showed bactericidal activity against nine of twelve clinical isolates studied. The bactericidal activity of the rabbit antiserum can be fully inhibited by the type B LOS but not the A or C LOS. These results indicate that the type B LOS-based conjugates can be used as vaccine components for further investigation

Biological and Immunological Characteristics of Lipooligosaccharide-Based Conjugate Vaccines for Serotype C Moraxella catarrhalis▿

Moraxella catarrhalis is an important bacterial cause of otitis media in children and respiratory tract infections in the elderly. Lipooligosaccharide (LOS), a major surface antigen of this bacterium, is a potential vaccine component against the organism. There are three major LOS serotypes (serotypes A, B, and C) in clinical isolates of M. catarrhalis. Our previous studies demonstrated that serotype A and B LOS-based conjugates were immunogenic in animals and elicited bactericidal antibodies. In this study, LOS from serotype C strain 26404 was isolated, detoxified, and conjugated to tetanus toxoid (TT) or the cross-reactive mutant (CRM) of diphtheria toxin to form detoxified LOS (dLOS)-TT, dLOS-CRM-1, and dLOS-CRM-2 vaccine candidates. The molar ratios (dLOS/protein) of the resulting conjugates were 47:1, 19:1, and 32:1, respectively, while the weight ratios were 0.94, 0.84 and 1.44, respectively. All conjugates were highly immunogenic in both mouse and rabbit models. Three subcutaneous injections of each conjugate formulated with the Ribi adjuvant elicited >700-fold increases in serum anti-LOS immunoglobulin G levels in mice (5 μg of dLOS) and >2,000-fold increases in rabbits (50 μg of dLOS). The resulting mouse and rabbit antisera showed complement-mediated bactericidal activity against the homologous strain. In addition, a representative rabbit antiserum showed bactericidal activity against 14 of 18 testable strains, and this bactericidal activity could be 100% inhibited by the serotype C or A LOS but only 30% inhibited by the serotype B LOS. These results indicate that the serotype C LOS-based conjugates can be used as vaccine components for further investigation in humans

Intranasal immunization of the combined lipooligosaccharide conjugates protects mice from the challenges with three serotypes of Moraxella catarrhalis.

There are no licensed vaccines available against Moraxella catarrhalis, a significant human respiratory pathogen. Lipooligosaccharide (LOS) based conjugate vaccines derived from individual serotype M. catarrhalis only showed partial protection coverage. A vaccine combining LOS conjugates of two or three serotypes might provide a broader protection.Mice were immunized intranasally with the combined conjugates consisting of LOS from serotype A and B or serotype A, B, and C followed by challenge with different M. catarrhalis strains of three serotypes. Mouse lungs, nasal washes, and sera were collected after each challenge for bacterial counts, histological evaluation, cytokine profiles, antibody level and binding activity determinations.Intranasal administration of the combined LOS conjugates not only enhanced pulmonary bacterial clearance of all three serotypes of M. catarrhalis strains in vaccinated mice, but also elevated serotype-specific anti-LOS immunoglobulin (Ig)A and IgG titers in nasal wash and serum respectively. Mice vaccinated with the combined LOS conjugates also showed increased interferon (IFN)-γ, interleukin (IL)-12, and IL-4 in the lungs after challenges. Compared to the control group, mice immunized with the combined LOS conjugates also showed reduced lung inflammation after M. catarrhalis infections. The hyperimmune sera induced by the combined conjugates exhibited a broad cross-reactivity toward all three serotypes of M. catarrhalis under transmission electron microscopy.The combined vaccine of serotype A and B LOS conjugates provides protection against most M. catarrhalis strains by eliciting humoral and cellular immune responses

PIVKA-II level is correlated to development of portal vein tumor thrombus in patients with HBV-related hepatocellular carcinoma

Abstract Aim To evaluate the correlation of serum PIVKA-II levels and development of portal vein tumor thrombus (PVTT) in hepatocellular carcinoma (HCC) patients. Methods One hundred and twenty-three patients with newly diagnosed HCC were included in this study between March 2016 and October 2018. Thirty-five of these patients were detected with PVTT and all subjects were randomly divided to analysis group (N = 73) and validation (N = 50) group. Serum levels of PIVKA-II, laboratory tests including serum aspartate aminotransferase, total bilirubin, platelet count, albumin levels were demonstrated in all the patients. T-test, chi-squared test and logistic regression was used for analyzing data. Diagnostic efficiency and cut-off value of PIVKA-II in PVTT development of HCC patients were calculated using receiver operator curve (ROC) analysis. Results Serum level of PIVKA-II in HCC patients with PVTT was significantly higher than that in HCC patients without PVTT (995.8 mAU/ml vs 94.87 mAU/ml; P = 0.003), as well as D-dimer levels (2.12 mg/L vs 0.56 mg/L P = 0.001). Univariate analysis showed that high serum D-dimer level was an independent risk factor for development of PVTT (OR = 1.22, 95%CI 1.02–1.45). ROC curve showed that among analysis group, the area under ROC curve (AUROC) of PIVKA-II was 0.73 (95%CI 0.59–0.86). For the detection of PVTT in HCC, PIVKA-II had a sensitivity of 83.7% and a specificity of 69.2% at a cutoff of 221.26 mAU/ml, which had a sensitivity of 85.71% and a specificity of 55.56% in validation group, respectively. Conclusion Serum PIVKA-II level is a potential marker for diagnosis of PVTT in HCC patients, which may guide therapeutic strategy and assessment of tumor prognosis of HCC

Distinct GAB2 signaling pathways are essential for myeloid and lymphoid transformation and leukemogenesis by BCR-ABL1.

Tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1, the product of the Philadelphia (Ph) chromosome, have revolutionized treatment of patients with chronic myeloid leukemia (CML). However, acquired resistance to TKIs is a significant clinical problem in CML, and TKI therapy is much less effective against Ph(+)B-cell acute lymphoblastic leukemia (B-ALL). BCR-ABL1, via phosphorylated Tyr177, recruits the adapter GRB2-associated binding protein 2 (GAB2) as part of a GRB2/GAB2 complex. We showed previously that GAB2 is essential for BCR-ABL1-evoked myeloid transformation in vitro. Using a genetic strategy and mouse models of CML and B-ALL, we show here that GAB2 is essential for myeloid and lymphoid leukemogenesis by BCR-ABL1. In the mouse model, recipients of BCR-ABL1-transducedGab2(-/-)bone marrow failed to develop CML-like myeloproliferative neoplasia. Leukemogenesis was restored by expression of GAB2 but not by GAB2 mutants lacking binding sites for its effectors phosphatidylinositol 3-kinase (PI3K) or SRC homology 2-containing phosphotyrosine phosphatase 2 (SHP2). GAB2 deficiency also attenuated BCR-ABL1-induced B-ALL, but only the SHP2 binding site was required. The SHP2 and PI3K binding sites were differentially required for signaling downstream of GAB2. Hence, GAB2 transmits critical transforming signals from Tyr177 to PI3K and SHP2 for CML pathogenesis, whereas only the GAB2-SHP2 pathway is essential for lymphoid leukemogenesis. Given that GAB2 is dispensable for normal hematopoiesis, GAB2 and its effectors PI3K and SHP2 represent promising targets for therapy in Ph(+)hematologic neoplasms