Deregulation of the RAS GTPase cycle due to mutations in the three RAS genes is commonly associated with cancer development. Protein tyrosine phosphatase SHP2 promotes RAF-to-MAPK signaling pathway and is an essential factor in RAS-driven oncogenesis. Despite the emergence of SHP2 inhibitors for the treatment of cancers harbouring mutant KRAS, the mechanism underlying SHP2 activation of KRAS signaling remains unclear. Here we report tyrosyl-phosphorylation of endogenous RAS and demonstrate that KRAS phosphorylation via Src on Tyr32 and Tyr64 alters the conformation of switch I and II regions, which stalls multiple steps of the GTPase cycle and impairs binding to effectors. In contrast, SHP2 dephosphorylates KRAS, a process that is required to maintain dynamic canonical KRAS GTPase cycle. Notably, Src- and SHP2-mediated regulation of KRAS activity extends to oncogenic KRAS and the inhibition of SHP2 disrupts the phosphorylation cycle, shifting the equilibrium of the GTPase cycle towards the stalled ‘dark state’.

Survival for high-risk neuroblastoma remains poor and treatment for relapsed disease rarely leads to long-term cures. Large sequencing studies of neuroblastoma tumors from diagnosis have not identified common targetable driver mutations other than the 10% of tumors that harbor mutations in the anaplastic lymphoma kinase (ALK) gene. However, at neuroblastoma recurrence, more frequent mutations in genes in the RAS-MAPK pathway have been detected. The PTPN11-encoded tyrosine phosphatase SHP2 is an activator of the RAS pathway, and we and others have shown that pharmacologic inhibition of SHP2 suppresses the growth of various tumor types harboring KRAS mutations such as pancreatic and lung cancers. Here we report inhibition of growth and downstream RAS-MAPK signaling in neuroblastoma cells in response to treatment with the SHP2 inhibitors SHP099, II-B08, and RMC-4550. However, neuroblastoma cell lines harboring endogenous NRAS Q61K mutation (which is commonly detected at relapse) or isogenic neuroblastoma cells engineered to overexpress NRAS Q61K were distinctly resistant to SHP2 inhibitors. Combinations of SHP2 inhibitors with other RAS pathway inhibitors such as trametinib, vemurafenib, and ulixertinib were synergistic and reversed resistance to SHP2 inhibition in neuroblastoma in vitro and in vivo. These results suggest for the first time that combination therapies targeting SHP2 and other components of the RAS-MAPK pathway may be effective against conventional therapy-resistant relapsed neuroblastoma, including those that have acquired NRAS mutations.Significance: These findings suggest that conventional therapyresistant, relapsed neuroblastoma may be effectively treated via combined inhibition of SHP2 and MEK or ERK of the RAS-MAPK pathway.

Background: TAZ functions as a co-activator by up-regulating downstream transcriptional targets. Results: TAZ can negatively regulate transcription of many genes such as ⌬Np63 through TEAD transcription factor. Conclusion: TAZ can also function as a transcriptional co-repressor. Significance: Our findings open a new avenue for TAZ function in cancer.

Cancer cells bearing distinct KRAS mutations exhibit variable sensitivity to SHP2 inhibitors (SHP2i). Here we show that cells harboring KRAS Q61H are uniquely resistant to SHP2i, and investigate the underlying mechanisms using biophysics, molecular dynamics, and cell-based approaches. Q61H mutation impairs intrinsic and GAP-mediated GTP hydrolysis, and impedes activation by SOS1, but does not alter tyrosyl phosphorylation. Wild-type and Q61H-mutant KRAS are both phosphorylated by Src on Tyr32 and Tyr64 and dephosphorylated by SHP2, however, SHP2i does not reduce ERK phosphorylation in KRAS Q61H cells. Phosphorylation of wild-type and Gly12-mutant KRAS, which are associated with sensitivity to SHP2i, confers resistance to regulation by GAP and GEF activities and impairs binding to RAF, whereas the near-complete GAP/GEF-resistance of KRAS Q61H remains unaltered, and high-affinity RAF interaction is retained. SHP2 can stimulate KRAS signaling by modulating GEF/GAP activities and dephosphorylating KRAS, processes that fail to regulate signaling of the Q61H mutant.

Survival for high-risk neuroblastoma remains poor. Most patients who recur, present with metastatic disease, and few targetable pathways that govern spread to distant sites are currently known. We previously developed a metastatic mouse model to select cells with enhanced ability to spread to the bone and brain and identified a signature based on differentially expressed genes, which also predicted patient survival. To discover new neuroblastoma therapies, we utilized the Connectivity Map to identify compounds that can reverse this metastatic transcriptional signature and found calcipotriol, a vitamin D3 analog, to be a compound that selectively targets cell lines with enhanced metastatic potential. Calcipotriol treatment of enhanced metastatic, but not parental, cells reduces proliferation and survival via vitamin D receptor (VDR) signaling, increases the expression of RASSF2, a negative regulator of the Hippo signaling pathway, and reduces the levels of the Hippo pathway effectors YAP and TAZ. RASSF2 is required for the effects of calcipotriol and for the reduction of levels and nuclear localization of YAP/TAZ. Migration of the enhanced metastatic cells and YAP/TAZ levels are reduced after calcipotriol treatment and YAP overexpression reduces calcipotriol sensitivity. Furthermore, metastatic cells that overexpress VDR also showed lower tumor burden in vivo. Implications: This newly identified link between VDR signaling and the Hippo pathway could inform treatment strategies for metastatic neuroblastoma.

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Supplementary Figure from Vitamin D Receptor Activation Attenuates Hippo Pathway Effectors and Cell Survival in Metastatic Neuroblastoma

Supplementary Figure from Vitamin D Receptor Activation Attenuates Hippo Pathway Effectors and Cell Survival in Metastatic Neuroblastoma