Identification of novel therapeutic targets in acute leukemias with NRAS mutations using a pharmacologic approach

Abstract

Oncogenic forms of NRAS are frequently associated with hematologic malignancies and other cancers, making them important therapeutic targets. Inhibition of individual downstream effector molecules (eg, RAF kinase) have been complicated by the rapid development of resistance or activation of bypass pathways. For the purpose of identifying novel targets in NRAS-transformed cells, we performed a chemical screen using mutant NRAS transformed Ba/F3 cells to identify compounds with selective cytotoxicity. One of the compounds identified, GNF-7, potently and selectively inhibited NRAS-dependent cells in preclinical models of acute myelogenous leukemia and acute lymphoblastic leukemia. Mechanistic analysis revealed that its effects were mediated in part through combined inhibition of ACK1/AKT and of mitogen-activated protein kinase kinase kinase kinase 2 (germinal center kinase). Similar to genetic synthetic lethal approaches, these results suggest that small molecule screens can be used to identity novel therapeutic targets in cells addicted to RAS oncogenes.

Cooperative Epigenetic Remodeling by TET2 Loss and NRAS Mutation Drives Myeloid Transformation and MEK Inhibitor Sensitivity

“…TET2-knockdown THP-1 cells showed higher level of pERK1/2, pAKT and pS6 upon GM-CSF stimulation (Figure S6N) and generated a higher number of colonies in GM-CSF supplemented methylcellulose media compared to control cells (Figure S6O), indicating similar hypersensitivity to GM-CSF in TET2-knockdown THP-1 cells. We next assessed the impact of TET2 knockdown on NRAS Q61L mutant OCI-AML3 cells and FLT3-ITD positive MOLM13 cells (Nonami et al, 2015; Quentmeier et al, 2003). Hairpin-transduced OCI-AML3 and MOLM13 cells demonstrated efficient knockdown of TET2 (Figure S6P and S6Q).…”

Inhibition of Wild-Type p53-Expressing AML by the Novel Small Molecule HDM2 Inhibitor CGM097

“…All cell lines matched >80% with lines listed in the DSMZ Cell Line Bank STR Profile Information. Other cell lines expressing mutated RAS or wild-type RAS were sequenced or tested with pharmacological inhibitors for validation of cell line integrity (24). All cell lines were obtained between 2000 and 2015.…”

“…Cell counts for proliferation studies were obtained using the trypan blue exclusion assay, as previously described (24). Error bars represent the standard error of the mean for each data point.…”

“…Error bars represent the standard error of the mean for each data point. Programmed cell death of inhibitor-treated cells was determined using the Annexin-V-Fluos Staining Kit (Boehringer Mannheim, Indianapolis, IN), as previously described (24). Cell cycle analysis was performed as previously described (24).…”

Synthetic lethality: emerging targets and opportunities in melanoma

“…Although there have been no studies assessing NRAS as a synthetic dose lethal target in melanoma, in acute myeloid leukaemia (AML) the multitarget kinase inhibitor GNF‐7 has been reported to induce apoptosis in NRAS ‐mutant but not wild‐type AML. Further mechanistic investigation using shRNA knockdown and a panel of kinase inhibitors suggest a possible synthetic dose lethal interaction between NRAS mutation and GCK, a serine/threonine kinase involved in the activation of JNK and MEKK1 (Nonami et al., ).…”