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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Article
  • Peer Reviewed
UC Irvine Previously Published Works (2001)
The catalytic activity of the c-Abl tyrosine kinase is tightly regulated by its Src homology 3 (SH3) domain through a complex mechanism that may involve intramolecular binding to Pro242 in the linker region between the SH2 and catalytic domains as well as interactions with a trans-inhibitor. We analysed the effect of mutation or replacement of SH3 on c-Abl tyrosine kinase activity and transformation. Random mutagenesis of SH3 identified several novel point mutations that dysregulated c-Abl kinase activity in vivo, but the RT loop was insensitive to mutational activation. Activating SH3 mutations abolished binding of proline-rich SH3 ligands in vitro, while mutations at Ser140 in the connector between the SH3 and SH2 domains activated Abl kinase activity in vivo and in vitro but did not impair SH3 ligand-binding. Abl was regulated efficiently when its SH3 domain was replaced with a heterologous SH3 from c-Src that binds a different spectrum of proline-rich ligands, but not by substitution of a modular WW domain with similar ligand-binding specificity. These results suggest that the SH3 domain regulates Abl principally by binding to the atypical intramolecular ligand Pro242 rather than a canonical PxxP ligand. Coordination between the SH3 and SH2 domains mediated by the connector region may be required for regulation of Abl even in the absence of SH2 ligand binding.
    Cover page: Mutational analysis of the regulatory function of the c-Abl Src homology 3 domainCreative Commons 'BY' version 4.0 license
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2001)
    The effect of mutations in the Src homology 2 (SH2) domain of the BCR/ABL oncogene on leukemogenesis was tested in a quantitative murine bone marrow transduction/transplantation assay that accurately models human Philadelphia-positive B-lymphoid leukemia and chronic myeloid leukemia (CML). The SH2 domain was not required for induction of B-lymphoid leukemia in mice by BCR/ABL. Under conditions where the p190 and p210 forms of BCR/ABL induce fatal CML-like myeloproliferative disease within 4 weeks, p210 SH2 mutants induced CML-like disease in some mice only after a significant delay, with other recipients succumbing to B-lymphoid leukemia instead. In contrast, p190 BCR/ABL SH2 point and deletion mutants rapidly induced CML-like disease. These results provide the first direct evidence of significant differences in cell signaling by the Bcr/Abl tyrosine kinase between these distinct leukemias. Contrary to previous observations, high levels of phosphatidylinositol 3-kinase (PI 3-kinase) activity in primary malignant lymphoblasts and myeloid cells from recipients of marrow transduced with the BCR/ABL SH2 mutants were found. Hence, the decreased induction of CML-like disease by the p210 BCR/ABL SH2 mutants is not due to impaired activation of PI 3-kinase.
      Cover page: The Src homology 2 domain of Bcr/Abl is required for efficient induction of chronic myeloid leukemia–like disease in mice but not for lymphoid leukemogenesis or activation of phosphatidylinositol 3-kinaseCreative Commons 'BY' version 4.0 license
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2004)

      A direct binding site for the Grb2 adapter protein is required for the induction of fatal chronic myeloid leukemia (CML)-like disease in mice by Bcr-Abl. Here, we demonstrate direct binding of Grb2 to the Tel-Abl (ETV6-Abl) fusion protein, the product of complex (9;12) chromosomal translocations in human leukemia, via tyrosine 314 encoded by TEL exon 5. A Tel-Abl point mutant (Y314F) and a splice variant without TEL exon 5 sequences (Deltae5) lacked Grb2 interaction and exhibited decreased binding and phosphorylation of the scaffolding protein Gab2 and impaired activation of phosphatidylinositol 3-kinase, Akt, and extracellular signal-regulated kinase/mitogen-activated protein kinase in hematopoietic cells. Tel-Abl Y314F and Deltae5 were unable to transform fibroblasts to anchorage-independent growth and were defective for B-lymphoid transformation in vitro and lymphoid leukemogenesis in vivo. Previously, we demonstrated that full-length Tel-Abl induced two distinct myeloproliferative diseases in mice: CML-like leukemia similar to that induced by Bcr-Abl and a novel syndrome of small-bowel myeloid infiltration endotoxemia and hepatic and renal failure. Lack of the Grb2 binding site had no effect on development of small bowel syndrome but significantly attenuated the induction of CML-like disease by Tel-Abl. These results suggest that direct binding of Grb2 is a common mechanism contributing to leukemogenesis by oncogenic Abl fusion proteins.

        Cover page: A direct binding site for Grb2 contributes to transformation and leukemogenesis by the Tel-Abl (ETV6-Abl) tyrosine kinase.
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2002)

        The Abl tyrosine kinase inhibitor STI-571 is effective therapy for stable phase chronic myeloid leukemia (CML) patients, but the majority of CML blast-crisis patients that respond to STI-571 relapse because of reactivation of Bcr-Abl signaling. Mutations of Thr-315 in the Abl kinase domain to Ile (T315I) were previously described in STI-571-resistant patients and likely cause resistance from steric interference with drug binding. Here we identify mutations of Tyr-253 in the nucleotide-binding (P) loop of the Abl kinase domain to Phe or His in patients with advanced CML and acquired STI-571 resistance. Bcr-Abl Y253F demonstrated intermediate resistance to STI-571 in vitro and in vivo when compared with Bcr-Abl T315I. The response of Abl proteins to STI-571 was influenced by the regulatory state of the kinase and by tyrosine phosphorylation. The sensitivity of purified c-Abl to STI-571 was increased by a dysregulating mutation (P112L) in the Src homology 3 domain of Abl but decreased by phosphorylation at the regulatory Tyr-393. In contrast, the Y253F mutation dysregulated c-Abl and conferred intrinsic but not absolute resistance to STI-571 that was independent of Tyr-393 phosphorylation. The Abl P-loop is a second target for mutations that confer resistance to STI-571 in advanced CML, and the Y253F mutation may impair the induced-fit interaction of STI-571 with the Abl catalytic domain rather than sterically blocking binding of the drug. Because clinical resistance induced by the Y253F mutation might be overcome by dose escalation of STI-571, molecular genotyping of STI-571-resistant patients may provide information useful for rational therapeutic management.

          Cover page: Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop.
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2004)
          8p11 myeloproliferative syndrome (EMS) is a hematopoietic stem cell disorder characterized by myeloid hyperplasia and non-Hodgkin's lymphoma with chromosomal translocations fusing several genes, most commonly ZNF198, to fibroblast growth factor receptor-1 (FGFR1). However, patients with BCR-FGFR1 fusion present with typical chronic myeloid leukemia (CML). We demonstrate that ZNF198-FGFR1 induces EMS-like disease in mice, with myeloproliferation and T lymphoma arising from common multipotential progenitors. Mutation of FGFR1 Tyr766 attenuates both myeloid and lymphoid diseases, identifying phospholipase C-gamma1 as a downstream effector. Bcr-FGFR1 binds Grb2 via Bcr Tyr177 and induces CML-like leukemia in mice, whereas Bcr-FGFR1/Y177F lacks Grb2 binding and causes EMS-like disease. These results implicate different signaling pathways originating from both kinase and fusion partner in the pathogenesis of CML and EMS.
            Cover page: Distinct stem cell myeloproliferative/T lymphoma syndromes induced by ZNF198-FGFR1 and BCR-FGFR1 fusion genes from 8p11 translocationsCreative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2020)

            Objectives

            The challenges of understanding how interventions influence follow-up medical care are magnified during genomic testing because few patients have received it to date and because the scope of information it provides is complex and often unexpected. We tested a novel strategy for quantifying downstream healthcare utilization after genomic testing to more comprehensively and efficiently identify related services. We also evaluated the effectiveness of different methods for collecting these data.

            Methods

            We developed a risk-based approach for a trial of newborn genomic sequencing in which we defined primary conditions based on existing diagnoses and family histories of disease and defined secondary conditions based on unexpected findings. We then created patient-specific lists of services associated with managing primary and secondary conditions. Services were quantified based on medical record reviews, surveys, and telephone check-ins with parents.

            Results

            By focusing on services that genomic testing would most likely influence in the short-term, we reduced the number of services in our analyses by more than 90% compared with analyses of all observed services. We also identified the same services that were ordered in response to unexpected findings as were identified during expert review and by confirming whether recommendations were completed. Data also showed that quantifying healthcare utilization with surveys and telephone check-ins alone would have missed the majority of attributable services.

            Conclusions

            Our risk-based strategy provides an improved approach for assessing the short-term impact of genomic testing and other interventions on healthcare utilization while conforming as much as possible to existing best-practice recommendations.
              Cover page: Quantifying Downstream Healthcare Utilization in Studies of Genomic Testing
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