Myelopoiesis Requires a Noncatalytic, Ras-Independent Function of SHP-2.

Activating mutations in PTPN11 , which encodes the tyrosine phosphatase SHP-2, comprise the most frequent genetic lesion in juvenile myelomonocytic leukemia (JMML). Other etiologies of JMML include activating mutations in NRAS or KRAS2 and inactivation of the tumor suppressor NF1 . These and other observations imply that PTPN11 functions in a common genetic pathway with RAS and NF1 . Ras proteins are signal switch molecules that respond to extracellular stimuli by cycling between inactive GDP-bound and active GTP-bound conformations. Oncogenic alleles encode proteins that preferentially accumulate in the GTP-bound form. While NF1 encodes a GTPase activating protein for Ras that directly modulates Ras-GTP levels, the biochemical relationship between SHP-2 phosphatase activity and Ras signaling remains unclear. Most mammalian systems place SHP-2 upstream of Ras activation, but the mechanism is not known. Studies of Ptpn11 mutant embryos and of chimeric mice have shown that SHP-2 plays an essential role in hematopoietic development. We tested the hypothesis that the essential function of SHP-2 in primary hematopoietic cells is to activate Ras. To do this, we determined if Ras activation by expression of an oncogenic Kras2 allele could eliminate the requirement for SHP-2. We used conditional alleles of Kras2 ( LSL-Kras G12D ) and Ptpn11 ( Ptpn11 flox/flox ) coupled with the inducible Mx1-Cre transgene. Juvenile mice were injected with polyI:polyC, resulting in expression of K-Ras G12D and inactivation of Ptpn11 . Although these mice uniformly developed fatal MPD similar to what we previously reported in Mx1-Cre , LSL-Kras G12D mice (Braun et al., PNAS 101(2):597–602), myeloid progenitors invariably retained an intact Ptpn11 allele despite uniform activation of the conditional Kras G12D allele. These data suggested that there was strong selective pressure to retain a functional Ptpn11 allele despite oncogenic K-Ras expression. To test this hypothesis directly, we enumerated myeloid progenitor colonies in methylcellulose medium immediately after inactivating Ptpn11 and activating Kras G12D via retroviral transduction. This confirmed a strong dependence on SHP-2 for formation of myeloid colonies either in the presence or absence of Kras G12D . Infecting Ptpn11 flox/flox , LSL-Kras G12D cells with a Ptpn11 -IRES- Cre virus fully restored the aberrant growth phenotype of Kras G12D mutant cells. Remarkably, alleles encoding phosphatase-deficient SHP-2 proteins also rescued CFU-GM growth. These data indicate that SHP-2 is required for growth of both normal and neoplastic myeloid progenitors in vivo and in vitro . Our data support a model in which SHP-2 has essential hematopoietic functions that are independent of Ras activation and do not require SHP-2 phosphatase activity. The presence of protein-protein interaction domains in SHP-2 suggests that it may have a noncatalytic adaptor function. Because transformation by leukemogenic Ptpn11 alleles requires catalytic activity, our data imply that inhibition of SHP-2 catalysis will selectively target neoplastic hematopoietic progenitors.