Constitutive Ras Signaling Research Articles

Abstract 5520: Oncogenic RTK signaling inhibits Spred1/NF1 to sustain constitutive Ras/MAPK signaling

Abstract Spred proteins negatively regulate Ras/MAPK signaling following growth factor stimulation. Inhibition of Ras primary occurs through Spreds ability to bind and localize NF1, a RasGAP and major tumor suppressor, to the plasma membrane. Spred1 and NF1 loss-of-function mutations occur across multiple cancer types including non-small cell lung carcinoma, glioblastoma, melanoma, stomach carcinoma, and uterine carcinosarcoma. Here we demonstrate that oncogenic RTK signaling leads to phosphorylation of Spred1(S105) in the EVH domain, which disrupts Spred1-NF1 binding and function. Phosphomimetic Spred1 is unable to suppress Ras-GTP following growth factor stimulation and cancer cell proliferation. The Spred1(S105) kinase is likely to be a CDK based on in vitro kinase and in vivo cell line assays. Our findings provide one potential mechanism by which oncogenic RTK signaling disrupts negative feedback to sustain constitutive Ras signaling. Furthermore, this work may elucidate a novel therapeutic target for restoring NF1-mediated inhibition of Ras. Citation Format: Evan Markegard, Ellen L. Mercado, Pau Castel, Jillian Silva, Jacqueline Galeas, Kathy Li, Anatoly Urisman, Frank McCormick. Oncogenic RTK signaling inhibits Spred1/NF1 to sustain constitutive Ras/MAPK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5520.

Constitutive Ras signaling and Ink4a/Arf inactivation cooperate during the development of B-ALL in mice.

Despite recent advances in treatment, human precursor B-cell acute lymphoblastic leukemia (B-ALL) remains a challenging clinical entity. Recent genome-wide studies have uncovered frequent genetic alterations involving RAS pathway mutations and loss of the INK4A/ARF locus, suggesting their important role in the pathogenesis, relapse, and chemotherapy resistance of B-ALL. To better understand the oncogenic mechanisms by which these alterations might promote B-ALL and to develop an in vivo preclinical model of relapsed B-ALL, we engineered mouse strains with induced somatic KrasG12D pathway activation and/or loss of Ink4a/Arf during early stages of B-cell development. Although constitutive activation of KrasG12D in B cells induced prominent transcriptional changes that resulted in enhanced proliferation, it was not sufficient by itself to induce development of a high-grade leukemia/lymphoma. Instead, in 40% of mice, these engineered mutations promoted development of a clonal low-grade lymphoproliferative disorder resembling human extranodal marginal-zone lymphoma of mucosa-associated lymphoid tissue or lymphoplasmacytic lymphoma. Interestingly, loss of the Ink4a/Arf locus, apart from reducing the number of apoptotic B cells broadly attenuated KrasG12D-induced transcriptional signatures. However, combined Kras activation and Ink4a/Arf inactivation cooperated functionally to induce a fully penetrant, highly aggressive B-ALL phenotype resembling high-risk subtypes of human B-ALL such as BCR-ABL and CRFL2-rearranged. Ninety percent of examined murine B-ALL tumors showed loss of the wild-type Ink4a/Arf locus without acquisition of highly recurrent cooperating events, underscoring the role of Ink4a/Arf in restraining Kras-driven oncogenesis in the lymphoid compartment. These data highlight the importance of functional cooperation between mutated Kras and Ink4a/Arf loss on B-ALL.

Abstract 1370: EGFR-mediated Spred1 phosphorylation inhibits NF1 to sustain constitutive Ras/MAPK signaling

Abstract Spred proteins negatively regulate Ras/MAPK signaling following growth factor stimulation. Inhibition of Ras primary occurs through Spreds ability to bind and localize NF1, a RasGAP and major tumor suppressor, to the plasma membrane. Spred1 and NF1 loss-of-function mutations occur across multiple cancer types including non-small cell lung carcinoma, glioblastoma, melanoma, stomach carcinoma, and uterine carcinosarcoma. Here we demonstrate that EGFR signaling disrupts Spred1-NF1 binding. Mass spectrometry was performed on cells overexpressing EGFRL858R to identify potential phosphorylation sites on Spred1 and NF1 that could disrupt Spred1-NF1 binding by steric hindrance. A serine phosphorylation site on Spred1 was identified in which a phosphomimetic and phosphodeficient mutant decreased or increased Spred1-NF1 binding, respectively. Phosphomimetic Spred1 is unable to suppress Ras-GTP following EGF stimulation. Therefore, phosphorylation of Spred1 at this site by a serine kinase downstream of EGFR may disrupt Spred1-NF1 binding. To identify the Spred1 kinase we are performing an in vitro kinase assay and an unbiased CRISPRa screen. Our findings provide one potential mechanism by which EGFR signaling disrupts negative feedback to sustain constitutive Ras signaling. Furthermore, this work may elucidate a novel therapeutic target for restoring NF1-mediated inhibition of Ras. Citation Format: Evan Markegard, Ellen L. Mercado, Jillian M. Silva, Jacqueline Galeas, Marena I. Trinidad, Anatoly Urisman, Frank McCormick. EGFR-mediated Spred1 phosphorylation inhibits NF1 to sustain constitutive Ras/MAPK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1370. doi:10.1158/1538-7445.AM2017-1370

Abstract 1874: Oncogenic EGFR signaling inhibits the Spred1-NF1 interaction to sustain constitutive Ras signaling

Abstract Spred proteins negatively regulate Ras/MAPK signaling following mitogen stimulation. Inhibition of Ras primary occurs through Spreds ability to bind and localize NF1, a RasGAP and major tumor suppressor, to the plasma membrane. Loss-of-function Spred1 and NF1 mutations occur across multiple cancer types including melanoma, non-small cell lung carcinoma, stomach carcinoma, and uterine carcinosarcoma. Here we demonstrate that oncogenic EGFR signaling disrupts Spred1-NF1 binding. Mass spectrometry was performed on cells overexpressing EGFRL858R to identify potential phosphorylation sites on Spred1 and NF1 that could disrupt Spred1-NF1 binding by steric hindrance. A serine phosphorylation site on Spred1 was identified in which a phosphomimetic and phosphodeficient mutant decreased or increased Spred1-NF1 binding, respectively. Therefore, phosphorylation of Spred1 at this site by a serine kinase downstream of oncogenic EGFR may disrupt Spred1-NF1 binding. Our findings provide one potential mechanism by which oncogenic EGFR signaling disrupts negative feedback to allow for constitutive Ras signaling. Furthermore, this work may elucidate a novel kinase therapeutic target for restoring NF1 mediated inhibition of Ras. Citation Format: Evan Markegard, Ellen L. Mercado, Jacqueline Galeas, Marena I. Trinidad, Anatoly Urisman, Frank McCormick. Oncogenic EGFR signaling inhibits the Spred1-NF1 interaction to sustain constitutive Ras signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1874.

Primary murine CD4+ T cells fail to acquire the ability to produce effector cytokines when active Ras is present during Th1/Th2 differentiation.

Constitutive Ras signaling has been shown to augment IL-2 production, reverse anergy, and functionally replace many aspects of CD28 co-stimulation in CD4+ T cells. These data raise the possibility that introduction of active Ras into primary T cells might result in improved functionality in pathologic situations of T cell dysfunction, such as cancer or chronic viral infection. To test the biologic effects of active Ras in primary T cells, CD4+ T cells from Coxsackie-Adenovirus Receptor Transgenic mice were transduced with an adenovirus encoding active Ras. As expected, active Ras augmented IL-2 production in naive CD4+ T cells. However, when cells were cultured for 4 days under conditions to promote effector cell differentiation, active Ras inhibited the ability of CD4+ T cells to acquire a Th1 or Th2 effector cytokine profile. This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression. Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus. Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4+ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci. Alternative strategies to potentiate Ras pathway signaling in T cells in a more regulated fashion should be considered as a therapeutic approach to improve immune responses in vivo.

Regulation of Drosophila Metamorphosis by Xenobiotic Response Regulators

Mammalian Nrf2-Keap1 and the homologous Drosophila CncC-dKeap1 protein complexes regulate both transcriptional responses to xenobiotic compounds as well as native cellular and developmental processes. The relationships between the functions of these proteins in xenobiotic responses and in development were unknown. We investigated the genes regulated by CncC and dKeap1 during development and the signal transduction pathways that modulate their functions. CncC and dKeap1 were enriched within the nuclei in many tissues, in contrast to the reported cytoplasmic localization of Keap1 and Nrf2 in cultured mammalian cells. CncC and dKeap1 occupied ecdysone-regulated early puffs on polytene chromosomes. Depletion of either CncC or dKeap1 in salivary glands selectively reduced early puff gene transcription. CncC and dKeap1 depletion in the prothoracic gland as well as cncCK6/K6 and dKeap1EY5/EY5 loss of function mutations in embryos reduced ecdysone-biosynthetic gene transcription. In contrast, dKeap1 depletion and the dKeap1EY5/EY5 loss of function mutation enhanced xenobiotic response gene transcription in larvae and embryos, respectively. Depletion of CncC or dKeap1 in the prothoracic gland delayed pupation by decreasing larval ecdysteroid levels. CncC depletion suppressed the premature pupation and developmental arrest caused by constitutive Ras signaling in the prothoracic gland; conversely, constitutive Ras signaling altered the loci occupied by CncC on polytene chromosomes and activated transcription of genes at these loci. The effects of CncC and dKeap1 on both ecdysone-biosynthetic and ecdysone-regulated gene transcription, and the roles of CncC in Ras signaling in the prothoracic gland, establish the functions of these proteins in the neuroendocrine axis that coordinates insect metamorphosis.

Evidence Implicating the Ras Pathway in Multiple CD28 Costimulatory Functions in CD4+ T Cells

CD28 costimulation is a critical event in the full activation of CD4+ T cells that augments cytokine gene transcription, promotes cytokine mRNA stability, prevents induction of anergy, increases cellular metabolism, and increases cell survival. However, despite extensive biochemical analysis of the signaling events downstream of CD28, molecular pathways sufficient to functionally replace the diverse aspects of CD28-mediated costimulation in normal T cells have not been identified. Ras/MAPK signaling is a critical pathway downstream of T cell receptor stimulation, but its role in CD28-mediated costimulation has been controversial. We observed that physiologic CD28 costimulation caused a relocalization of the RasGEF RasGRP to the T cell-APC interface by confocal microscopy. In whole cell biochemical analysis, CD28 cross-linking with either anti-CD28 antibody or B7.1-Ig augmented TCR-induced Ras activation. To determine whether Ras signaling was sufficient to functionally mimic CD28 costimulation, we utilized an adenoviral vector encoding constitutively active H-Ras (61L) to transduce normal, Coxsackie-Adenovirus Receptor (CAR) transgenic CD4+ T cells. Like costimulation via CD28, active Ras induced AKT, JNK and ERK phosphorylation. In addition, constitutive Ras signaling mimicked the ability of CD28 to costimulate IL-2 protein secretion, prevent anergy induction, increase glucose uptake, and promote cell survival. Importantly, we also found that active Ras mimicked the mechanism by which CD28 costimulates IL-2 production: by increasing IL-2 gene transcription, and promoting IL-2 mRNA stability. Finally, active Ras was able to induce IL-2 production when combined with ionomycin stimulation in a MEK-1-dependent fashion. Our results are consistent with a central role for Ras signaling in CD28-mediated costimulation.

Sporadic Notch1 Mutations Found in Human T-ALL Drive T Cell Development but Not Leukemogenesis in Murine Models: Implications for Pathogenesis and gamma-Secretase Inhibitor (GSI) Therapy.

The presence of gain-of-function Notch1 mutations in more than 50% of human T-ALL patient samples is consistent with a central role for Notch1 signals in the pathogenesis of T-ALL (Weng et al., 2004, Science, 306, 269-71). Notch1 mutations are of two types:common mis-sense or insertional mutations in the extracellular heterodimerization (HD) domain that cause heightened proteolytic activation of Notch1 by the gamma-secretase protease complex, andless frequent frameshift or stop codon mutations that delete a C-terminal PEST destruction box and increase the duration of Notch1 signaling.To investigate the significance of these mutations in the pathogenesis of T-ALL, the HD mutations L1601P, L1594P, and P12-ins (which produce weak, moderate, and strong ligand-independent increases in Notch1 signaling, respectively; Malecki et al., 2006, Mol. Cell. Biol. 12, 4642–51), and a PEST deletion (which has little intrinsic effect on Notch1 signaling) were transduced into hematopoietic progenitor cells. These cells were then used to reconstitute lethally irradiated mice, which were monitored for aberrant lymphocyte development and leukemia. The PEST deletion by itself did not produce a phenotype, consistent with prior studies (Feldman et al., 2000, Blood, 96, 1906–13). The two weakest HD mutations, L1601P and L1594P, induced ectopic T development in the bone marrow and inhibited CD19+ B-lymphoid and Lin-Sca-1+Kit-CD16/32+CD34+ granulocyte-macrophage progenitor development, but failed to induce T-ALL after more than 6 months. In contrast, the strongest HD mutation, P12-ins, induced leukemia with a penetrance of ~40%. When non-leukemogenic HD mutations were paired in cis with a PEST deletion (an arrangement found in ~15% of T-ALL that results in synergistic increases in signal strength), T-ALL occurred in a minority of animals (median time, ~4 months post-transplantation). Together, these data show that the level of Notch1 signaling required for induction of leukemia is higher than that needed for fate-determination during hematopoiesis and that most solitary Notch mutations fail to achieve the level needed for efficient induction of T-ALL. To investigate whether weak Notch alleles cooperate with other oncogenes, the L1601P and L1601P/PEST alleles were retrovirally introduced into lymphoid progenitors engineered to have constitutive Ras signaling, which has been shown to induce T-ALL (Hawley et al, Oncogene, 1995, 11, 1113–23). The L1601P and L1601P/PEST alleles shortened the mean time to leukemic presentation from 91 days to 76 and 52 days, respectively, indicating that these weak Notch1 alleles can cooperate with Ras signals to enhance leukemogenesis. GSI abolished the growth of cell lines derived from these tumors, indicating a continuing dependency on Notch1 signals. In summary, these experiments suggest that a supraphysiologic level of Notch signaling is required for efficient induction of T-ALL, but that once other oncogenic events have occurred, lower doses suffice to promote leukemogenesis cooperatively. However, even when Notch1 mutations are not initiating events, tumors remain “Notch addicted” and can be targeted pharmacologically with GSI. These findings reinforce the notion that the Notch pathway is a rational therapeutic target in T-ALL.

STAT1-independent inhibition of cyclooxygenase-2 expression by IFNγ; a common pathway of IFNγ-mediated gene repression but not gene activation

Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of prostaglandins, promotes the development of colorectal cancer, and is a key molecular target of non-steroidal anti-inflammatory drugs, compounds that reduce the relative risk of developing colon cancer. In this study, we showed that interferon gamma (IFNgamma) inhibits the expression of COX-2 protein in intestinal epithelial cells (IECs) through a pathway that requires Janus-activated kinase (JAK) activity. In contrast, we demonstrated that transcriptional inhibition of COX-2 by IFNbeta or IFNgamma occurs in cells with silenced signal transducer and activator of transcription 1 (STAT1) expression and that IFNs retained the ability to inhibit COX-2 transcription in cells with activated RasV12, in which IFNgamma failed to induce STAT1. Thus, unlike the activity of JAK, STAT1 is not required for the inhibition of COX-2 expression by IFNgamma. In contrast to COX-2, the activation of genes in response to IFNgamma, such as interferon regulatory factor-1, was severely impaired by both STAT1 silencing and by constitutive Ras signaling. To determine whether there is a general differential requirement for STAT1 in gene activation and gene repression in response to IFNgamma in intestinal cells, we performed genome-wide analysis of IFNgamma target genes in an IEC line in which STAT1 expression was silenced by small interfering RNA. The results confirmed that the activation of the majority of genes by IFNgamma required STAT1. In contrast, the repression of several genes, as we showed for COX-2 specifically, was largely unaffected in cells with silenced STAT1. Our results therefore demonstrate that in general gene activation by IFNgamma is more sensitive to STAT1 deficiency than gene repression, and suggest that IFNgamma activates and represses gene expression via distinct pathways that can be distinguished, at least in part, by their requirement for STAT1.

Ras GTPase-activating protein gap1 of the homobasidiomycete Schizophyllum commune regulates hyphal growth orientation and sexual development.

The white rot fungus Schizophyllum commune is used for the analysis of mating and sexual development in homobasidiomycete fungi. In this study, we isolated the gene gap1 encoding a GTPase-activating protein for Ras. Disruption of gap1 should therefore lead to strains accumulating Ras in its activated, GTP-bound state and to constitutive Ras signaling. Haploid Deltagap1 monokaryons of different mating types did not show alterations in mating behavior in the four different mating interactions possible in fungi expressing a tetrapolar mating type system. Instead, the growth rate in Deltagap1 monokaryons was reduced by ca. 25% and ca. 50% in homozygous Deltagap1/Deltagap1 dikaryons. Monokaryons, as well as homozygous dikaryons, carrying the disrupted gap1 alleles exhibited a disorientated growth pattern. Dikaryons showed a strong phenotype during clamp formation since hook cells failed to fuse with the peg beside them. Instead, the dikaryotic character of the hyphae was rescued by fusion of the hooks with nearby developing branches. Deltagap1/Deltagap1 dikaryons formed increased numbers of fruitbody primordia, whereas the amount of fruitbodies was not raised. Mature fruitbodies formed no or abnormal gills. No production of spores could be observed. The results suggest Ras involvement in growth, clamp formation, and fruitbody development.

Apoptosis coincident with the differentiation of skeletal myoblasts is delayed by caspase 3 inhibition and abrogated by MEK-independent constitutive Ras signaling

We demonstrate that during 23A2 skeletal myoblast differentiation, between 30–35% of the population apoptose. Both differentiation and apoptosis are controlled by the variables of cell density and time and these variables are inversely related. In response to conditions that permit both differentiation and apoptosis of parental 23A2 myoblasts, myoblasts rendered differentiation-defective by constitutive Ras signaling (A2:H-Ras myoblasts) do not apoptose. This is not merely a consequence of their differentiation-defective phenotype since myoblasts rendered differentiation-defective by expression of E1A (A2:E1A myoblasts) still apoptose. Although signaling through MEK is important to the survival of proliferating parental 23A2 myoblasts, constitutive signaling through MEK is not responsible for the survival of A2:H-Ras myoblasts. Finally, we demonstrate that caspase 3 is activated and that pharmacological inhibition of caspase 3 activity delays apoptosis without affecting differentiation. Abrogating apoptosis without affecting differentiation could be a useful approach to improve the efficacy of myoblast transfer in the treatment of muscular dystrophies.

Apoptosis coincident with the differentiation of skeletal myoblasts is delayed by caspase 3 inhibition and abrogated by MEK-independent constitutive Ras signaling

We demonstrate that during 23A2 skeletal myoblast differentiation, between 30-35% of the population apoptose. Both differentiation and apoptosis are controlled by the variables of cell density and time and these variables are inversely related. In response to conditions that permit both differentiation and apoptosis of parental 23A2 myoblasts, myoblasts rendered differentiation-defective by constitutive Ras signaling (A2:H-Ras myoblasts) do not apoptose. This is not merely a consequence of their differentiation-defective phenotype since myoblasts rendered differentiation-defective by expression of E1A (A2:E1A myoblasts) still apoptose. Although signaling through MEK is important to the survival of proliferating parental 23A2 myoblasts, constitutive signaling through MEK is not responsible for the survival of A2:H-Ras myoblasts. Finally, we demonstrate that caspase 3 is activated and that pharmacological inhibition of caspase 3 activity delays apoptosis without affecting differentiation. Abrogating apoptosis without affecting differentiation could be a useful approach to improve the efficacy of myoblast transfer in the treatment of muscular dystrophies.