Dual gene activation and knockout screen reveals directional dependencies in genetic networks.

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 analyze 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 uncharacterized genes to well-studied pathways and identifies targets relevant for therapeutic intervention

Basal LAT-diacylglycerol-RasGRP1 Signals in T Cells Maintain TCRα Gene Expression

In contrast to the well-characterized T cell receptor (TCR) signaling pathways that induce genes that drive T cell development or polarization of naïve CD4 T cells into the diverse TH1, TH2, TH17 and Treg lineages, it is unclear what signals maintain specific gene expression in mature resting T cells. Resting T cells residing in peripheral lymphoid organs exhibit low-level constitutive signaling. Whereas tonic signals in B cells are known to be critical for survival, the roles of tonic signals in peripheral T cells are unknown. Here we demonstrate that constitutive signals in Jurkat T cell lines are transduced via the adapter molecule LAT and the Ras exchange factor RasGRP1 to maintain expression of TCRα mRNA and surface expression of the TCR/CD3 complex. Independent approaches of reducing basal activity through the LAT-diacylglycerol-RasGRP pathway led to reduced constitutive Ras-MEK-ERK signals and decreased TCRα mRNA and surface TCR expression in Jurkat cells. However, loss of TCR expression takes several days in these cell line experiments. In agreement with these in vitro approaches, inducible deletion of Lat in vivo results in reduced TCRα mRNA- and surface TCR- expression in a delayed temporal manner as well. Lastly, we demonstrate that loss of basal LAT-RasGRP signals appears to lead to silencing or repression of TCRα transcription. We postulate that basal LAT-diacylglycerol-RasGRP signals fulfill a regulatory function in peripheral T lymphocytes by maintaining proper gene expression programs

Oncogene-mediated inhibition of the Spred1 tumor suppressor

Spred proteins are tumor suppressors which negatively regulate Ras/MAPK signaling following growth factor stimulation. Inhibition of Ras is thought to primarily occur through the ability of Spred1 to bind and localize Neurofibromin (NF1), a RasGAP, to the plasma membrane. SPRED1 and NF1 loss-of-function mutations tend to be mutually exclusive and occur across multiple cancer types and developmental diseases. We show oncogenic EGFRL858R signaling leads to the phosphorylation of Spred1 on serine 105 which disrupts the Spred1-Neurofibromin complex. The primary Spred1(S105) kinase was identified as CDK1. Spred1 directly binds CDK1 and enters the nucleus though a newly identified Class I nuclear localization sequence. Additionally, phosphomimetic Spred1 is unable to suppress Ras-GTP following growth factor stimulation and proliferation in the K562 leukemia cell line. Our findings provide one potential mechanism by which oncogenic signaling disrupts Spred1-Neurofibromin negative feedback of the Ras/MAPK pathway

Basal LAT-diacylglycerol-RasGRP1 signals in T cells maintain TCRα gene expression.

In contrast to the well-characterized T cell receptor (TCR) signaling pathways that induce genes that drive T cell development or polarization of naïve CD4 T cells into the diverse T(H)1, T(H)2, T(H)17 and T(reg) lineages, it is unclear what signals maintain specific gene expression in mature resting T cells. Resting T cells residing in peripheral lymphoid organs exhibit low-level constitutive signaling. Whereas tonic signals in B cells are known to be critical for survival, the roles of tonic signals in peripheral T cells are unknown. Here we demonstrate that constitutive signals in Jurkat T cell lines are transduced via the adapter molecule LAT and the Ras exchange factor RasGRP1 to maintain expression of TCRα mRNA and surface expression of the TCR/CD3 complex. Independent approaches of reducing basal activity through the LAT-diacylglycerol-RasGRP pathway led to reduced constitutive Ras-MEK-ERK signals and decreased TCRα mRNA and surface TCR expression in Jurkat cells. However, loss of TCR expression takes several days in these cell line experiments. In agreement with these in vitro approaches, inducible deletion of Lat in vivo results in reduced TCRα mRNA- and surface TCR-expression in a delayed temporal manner as well. Lastly, we demonstrate that loss of basal LAT-RasGRP signals appears to lead to silencing or repression of TCRα transcription. We postulate that basal LAT-diacylglycerol-RasGRP signals fulfill a regulatory function in peripheral T lymphocytes by maintaining proper gene expression programs

Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR.

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

Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR.

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

Preclinical efficacy of a RAF inhibitor that evades paradoxical MAPK pathway activation in protein kinase BRAF-mutant lung cancer

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 BRAFV600E-mutant allele, the spectrum of BRAF mutations in LA includes BRAFV600E (∼60% of cases) and non-V600E mutant alleles (∼40% of cases) such as BRAFG469A and BRAFG466V The presence of BRAFV600E in LA has prompted clinical trials testing selective BRAF inhibitors such as vemurafenib in BRAFV600E-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 BRAFV600E 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 BRAFV600E 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

Dual gene activation and knockout screen reveals directional dependencies in genetic networks.

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 analyze 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 uncharacterized genes to well-studied pathways and identifies targets relevant for therapeutic intervention