Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyse the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair. Based on the results from over 100,000 perturbed gene pairs, we reconstruct a directional dependency network for human K562 leukemia cells and demonstrate how our approach allows the determination of directionality in activating genetic interactions. Our interaction network connects previously uncharacterised genes to well-studied pathways and identifies targets relevant for therapeutic intervention.
Oncogenic activation of protein kinase BRAF drives tumor growth by promoting mitogen-activated protein kinase (MAPK) pathway signaling. Because oncogenic mutations in BRAF occur in ∼2-7% of lung adenocarcinoma (LA), BRAF-mutant LA is the most frequent cause of BRAF-mutant cancer mortality worldwide. Whereas most tumor types harbor predominantly the BRAF V600E -mutant allele, the spectrum of BRAF mutations in LA includes BRAF V600E (∼60% of cases) and non-V600E mutant alleles (∼40% of cases) such as BRAF G469A and BRAF G466V. The presence of BRAF V600E in LA has prompted clinical trials testing selective BRAF inhibitors such as vemurafenib in BRAF V600E -mutant patients. Despite promising clinical efficacy, both innate and acquired resistance often result from reactivation of MAPK pathway signaling, thus limiting durable responses to the current BRAF inhibitors. Further, the optimal therapeutic strategy to block non-V600E BRAF-mutant LA remains unclear. Here, we report the efficacy of the Raf proto-oncogene serine/ threonine protein kinase (RAF) inhibitor, PLX8394, that evades MAPK pathway reactivation in BRAF-mutant LA models. We show that PLX8394 treatment is effective in both BRAF V600E and certain non-V600 LA models, in vitro and in vivo. PLX8394 was effective against treatment-naive BRAF-mutant LAs and those with acquired vemurafenib resistance caused by an alternatively spliced, truncated BRAF V600E that promotes vemurafenib-insensitive MAPK pathway signaling. We further show that acquired PLX8394 resistance occurs via EGFR-mediated RAS-mTOR signaling and is prevented by upfront combination therapy with PLX8394 and either an EGFR or mTOR inhibitor. Our study provides a biological rationale and potential polytherapy strategy to aid the deployment of PLX8394 in lung cancer patients.ncogenic mutations in the BRAF serine/threonine protein kinase occur in a wide spectrum of solid tumor malignancies, most notably melanoma, colorectal cancer, and lung adenocarcinoma (1). Mutant BRAF promotes tumor growth by hyperactivating the RAF-MEK-ERK signaling cascade (2-5). BRAF V600E is the most common oncogenic form of BRAF in most tumor types, and selective RAF inhibitors such as vemurafenib and dabrafenib have demonstrated clinical efficacy in melanoma and LA harboring BRAF V600E (6-9). Despite these findings, ∼15% of BRAF mutant cancers do not respond to BRAF inhibitors, and the majority of patients who achieve a response will inevitably acquire resistance to these targeted agents, predominantly through reactivation of MAPK pathway signaling (4, 10-16).The clinical efficacy of RAF inhibitors depends on the degree of suppression of MAPK pathway output (8). Vemurafenib and dabrafenib paradoxically activate the MAPK pathway in cells with oncogenic RAS or increased upstream receptor signaling, thereby enhancing cellular proliferation that can promote cutaneous squamous cell carcinomas and keratoacanthomas that often harbor RAS mutations, and potentially other RAS-driven malignancies (17-25). Combination therapy with a MEK i...
SUMMARY Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions.
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