Biochemical and Functional Analysis of Ras Pathway Mutations in Myeloid Leukemia and Developmental Disorders

Noonan syndrome is a common dominant disorder characterized by short stature, facial dysmorphism, cardiac defects, and a predisposition to juvenile myelomonocytic leukemia (JMML). Germline PTPN11 mutations cause ~50% of Noonan syndrome. PTPN11 encodes SHP-2, a protein tyrosine phosphatase that relays signals from activated receptor complexes to Ras and other effectors. Studies of patient samples and in mouse models have demonstrated that hyperactive Ras signaling plays a central role in JMML. Based on the association of JMML with Noonan syndrome and the known role of SHP-2 in Ras signaling, our laboratory and others screened the PTPN11 gene in JMML, and discovered somatic mutations in ~ 35% of cases. I performed functional studies of mutant SHP-2 proteins associated with JMML and Noonan syndrome. I used primary murine hematopoietic cells to investigate the effects of mutant SHP-2 on proliferation, survival, and differentiation. The most common leukemia-associated amino acid substitution (E76K) induced a hypersensitive pattern of myeloid progenitor colony growth in response to granulocyte-macrophage colony-stimulating factor and interleukin 3 that was dependent on SHP-2 catalytic activity. E76K SHP-2 expression also enhanced the growth of immature progenitor cells, perturbed erythroid growth, and impaired normal differentiation. In addition, leukemia-associated SHP-2 mutations conferred stronger phenotypes in primary hematopoietic progenitors than a germline mutation found in individuals with Noonan syndrome. Although PTPN11 mutations account for 50% of Noonan syndrome, the genetic lesions in the remaining individuals were unknown. Our collaborators discovered de novo germline KRAS mutations that introduce V14I, T58I, or D153V amino acid substitutions in individuals with Noonan syndrome and P34R and F156L alterations in individuals with cardio-facio-cutaneous syndrome, which has overlapping phenotypic features with Noonan syndrome. I performed biochemical and functional analysis of these novel syndrome-associated K-Ras proteins. Mutant K-Ras proteins demonstrate a range of gain-of-function effects in different cell types, and biochemical analysis supports the idea that the intrinsic Ras guanosine nucleotide triphosphatase (GTPase) activity, the responsiveness of these proteins to GTPase activating proteins, and guanine nucleotide exchange all regulate developmental programs in vivo

Biochemical and Functional Characterization of Germ Line KRAS Mutations▿

Germ line missense mutations in HRAS and KRAS and in genes encoding molecules that function up- or downstream of Ras in cellular signaling networks cause a group of related developmental disorders that includes Costello syndrome, Noonan syndrome, and cardiofaciocutaneous syndrome. We performed detailed biochemical and functional studies of three mutant K-Ras proteins (P34R, D153V, and F156L) found in individuals with Noonan syndrome and cardiofaciocutaneous syndrome. Mutant K-Ras proteins demonstrate a range of gain-of-function effects in different cell types, and biochemical analysis supports the idea that the intrinsic Ras guanosine nucleotide triphosphatase (GTPase) activity, the responsiveness of these proteins to GTPase-activating proteins, and guanine nucleotide dissociation all regulate developmental programs in vivo

Functional analysis of leukemia-associated PTPN11 mutations in primary hematopoietic cells

PTPN11 encodes the protein tyrosine phosphatase SHP-2, which relays signals from growth factor receptors to Ras and other effectors. Germline PTPN11 mutations underlie about 50% of Noonan syndrome (NS), a developmental disorder that is associated with an elevated risk of juvenile myelomonocytic leukemia (JMML). Somatic PTPN11 mutations were recently identified in about 35% of patients with JMML; these mutations introduce amino acid substitutions that are largely distinct from those found in NS. We assessed the functional consequences of leukemia-associated PTPN11 mutations in murine hematopoietic cells. Expressing an E76K SHP-2 protein induced a hypersensitive pattern of granulocyte-macrophage colony-forming unit (CFU-GM) colony growth in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) that was dependent on SHP-2 catalytic activity. E76K SHP-2 expression also enhanced the growth of immature progenitor cells with high replating potential, perturbed erythroid growth, and impaired normal differentiation in liquid cultures. In addition, leukemia-associated SHP-2 mutations conferred a stronger phenotype than a germline mutation found in patients with NS. Mutant SHP-2 proteins induce aberrant growth in multiple hematopoietic compartments, which supports a primary role of hyperactive Ras in the pathogenesis of JMML

ALK positively regulates MYCN activity through repression of HBP1 expression

ALK mutations occur in 10% of primary neuroblastomas and represent a major target for precision treatment. In combination with MYCN amplification, ALK mutations infer an ultra-high-risk phenotype resulting in very poor patient prognosis. To open up opportunities for future precision drugging, a deeper understanding of the molecular consequences of constitutive ALK signaling and its relationship to MYCN activity in this aggressive pediatric tumor entity will be essential. We show that mutant ALK downregulates the ‘HMG-box transcription factor 1’ (HBP1) through the PI3K-AKT–FOXO3a signaling axis. HBP1 inhibits both the transcriptional activating and repressing activity of MYCN, the latter being mediated through PRC2 activity. HBP1 itself is under negative control of MYCN through miR-17~92. Combined targeting of HBP1 by PI3K antagonists and MYCN signaling by BET- or HDAC-inhibitors blocks MYCN activity and significantly reduces tumor growth, suggesting a novel targeted therapy option for high-risk neuroblastoma