Oncogenic Ras Requires SHP-2 To Induce Myeloproliferative Disease (MPD).
Authors: Braun Benjamin S. , Archard Joehleen , Yang Wentian , Chan Gordon , Neel Benjamin G. , Shannon Kevin M. 
Published: April 2020

Abstract
Oncogenic mutations in NRAS or KRAS2 are found in 20–40% of myeloid malignancies including acute myeloid leukemia, high risk subtypes of myelodysplastic syndrome, and in juvenile myelomonocytic leukemia (JMML) and other types of MPD. Mutant Ras proteins accumulate in the active, GTP-bound state due to impaired intrinsic GTPase activity and resistance to GTPase activating proteins (GAPs). Alternative genetic mechanisms, such as BCR-ABL fusion and inactivation of the NF1 tumor suppressor, also deregulate Ras signaling in myeloid malignancies. Moreover, the association of germline mutations in NF1 with a markedly increased risk of JMML and studies of KrasG12D and Nf1 mutant mice argue strongly that hyperactive Ras can initiate MPD in vivo. Activating mutations in PTPN11, which encodes the tyrosine phosphatase SHP-2, comprise the most frequent genetic lesion in JMML. As is the case for NF1, germline PTPN11 mutations impart an increased risk of developing JMML. These observations imply that PTPN11 functions in a common genetic pathway with RAS and NF1. While NF1 encodes a GAP for Ras that directly reduces Ras-GTP levels, data from various systems have shown that SHP-2 is a positive effector of Ras activation. However, exactly how depohsophorylation of SHP-2 substrates regulates Ras output and how other SHP-2 target proteins modulate its effects are incompletely understood. Studies of Ptpn11 mutant embryos and of chimeric mice have shown that SHP-2 plays an essential role in hematopoietic development. To test the hypothesis that Ras is strictly downstream of SHP-2 in primary hematopoietic cells, we used the conditional alleles LSL-KrasG12D and Ptpn11flox/flox coupled with the interferon-inducible Mx1-Cre transgene. Juvenile mice were injected with pI:pC, resulting in expression of K-RasG12D and inactivation of Ptpn11. These mice uniformly developed fatal MPD similar to what we previously reported in Mx1-Cre, LSL-KrasG12D mice (Braun et al., PNAS 101(2):597–602), although with slightly increased latency. Importantly, however, myeloid progenitors invariably retained at least one functional Ptpn11 allele despite uniform activation of the conditional KrasG12D allele. These data suggested that there was strong selective pressure to retain at least one functional Ptpn11 allele, even in the presence of oncogenic K-Ras expression. To address this hypothesis directly, we infected LSL-KrasG12D, Ptpn11flox/flox, and compound mutant fetal liver cells with a Cre-expressing retrovirus and enumerated myeloid progenitor colonies in methylcellulose medium. Remarkably, no granulocyte-macrophage colonies developed from Cre-expressing Ptpn11flox/flox fetal liver cells either in the presence or absence of KrasG12D. Infecting compound mutant cells with a Ptpn11-IRES-Cre virus fully restored the aberrant growth phenotype of KrasG12D mutant cells. 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 Ras-independent functions in hematopoiesis (i.e. that Ras is not strictly downstream of SHP-2). SHP-2 may be required either for efficient activation of canonical Ras effector pathways and/or to regulate signaling parallel to Ras. Our data imply that SHP-2 inhibition will ablate both normal and neoplastic hematopoietic progenitors. This may have a therapeutic role in preparing patients with JMML and other high risk MPDs for hematopoietic stem cell transplantation.