On the other hand, Lats2 knockdown, which was insufficient to affect TAZ expression, did not alter dasatinib sensitivity (Supplementary Figure 6)

On the other hand, Lats2 knockdown, which was insufficient to affect TAZ expression, did not alter dasatinib sensitivity (Supplementary Figure 6). Open in a separate window Figure 5 Dasatinib-induced senescence is usually mediated by Chk1, TAZ, and p21A. be used to treat KINSCLC. Our novel finding that dasatinib induced DNA damage and subsequently activated DNA repair pathways leading to senescence in KINSCLC cells represents a unique vulnerability with potential clinical applications. mutations, rearrangements, or translocations. However, only a minority of the remaining 80% of patients likely have targetable, activating kinase mutations or translocations, and there is a great need to identify additional effective therapies [1]. We previously recognized a patient with stage IV NSCLC harboring a novel mutation (Y472C) that experienced a near total radiographic response to the multitargeted kinase inhibitor dasatinib as the sole therapy; the patient lived without active malignancy for 7 years following treatment [2]. We discovered that Y472Cis usually a kinase-inactivating mutation (KIundergo senescence when exposed to dasatinib, whereas NSCLC with wild-type (WTand in patients [3]. The RAS/RAF/MEK/ERK pathway plays an important role in the progression of many human cancers. Once activated by surface receptors, RAS recruits RAF, a serine/threonine kinase, to the cell membrane and activates it. RAF then phosphorylates MEK, which in turn phosphorylates and activates ERK, leading to malignancy progression or senescence depending on the degree of ERK activation and crosstalk with other signaling pathways [4]. The 3 RAF proteins (A, B, and C) can form homodimers and heterodimers [5]. BRAF is usually by far the most frequently mutated isoform [6]. mutations can result in increased or decreased BRAF kinase activity, as well as kinase-neutral mutations, and mutations occur in 3C8% of patients with NSCLC [7C11] and many other tumor types [12]. KIstill paradoxically activates MEK/ERK to levels higher than those in cells with WTvia heterodimerization with CRAF (Raf-1) [13C16]. Similarly, inhibition of WTor expression of KIincreases CRAF-BRAF binding, activates CRAF, and enhances MEK/ERK activation [3, 14C16]. The underlying mechanism of dasatinib-induced senescence in KINSCLC cells is usually obscure. Dasatinib inhibits the activity of Src and Abl, as well as nearly 40 unique kinase targets [17, 18]. Dasatinib weakly inhibits BRAF, although only at concentrations higher than those needed to induce senescence, and it can induce BRAF-CRAF dimerization and CRAF activation in cells with activated RAS or KImutations [3, 19]. Although RAF dimerization was found to be necessary for dasatinib sensitivity, nilotinib, a kinase inhibitor with a similar kinase profile that also produced strong RAF dimerization, did not induce senescence. Another potent Src/Abl inhibitor, bosutinib, did not induce senescence [3]. Currently you will find no well-defined, canonical pathways that explain the observed dasatinib-induced senescence in KINSCLC cells. We sought to define the underlying mechanism leading to dasatinib-induced senescence in KINSCLC cells. We used 2 methods: gene expression arrays and reverse phase protein array (RPPA), in which we simultaneously examined the expression of 137 proteins and phosphoproteins in KIand WTNSCLC cell lines at baseline and following dasatinib treatment. Our approach was limited by the presence of only 2 NSCLC cell lines with endogenous KINSCLC cells. TAZ is usually part of the Hippo pathway that is a complex network of at least 35 proteins that converge on a core kinase cassette that consists of MST1/2, LATS1/2, SAV1, and MOB [20]. LATS1/2 phosphorylates the transcriptional co-activators YAP and TAZ that results in their ubiquitin-mediated proteolysis. TAZ has recently been defined as a novel oncogene in NSCLC cells where TAZ knock-down results in decreased anchorage-independent growth and tumor growth and WTNSCLC cells treated with dasatinib We used gene expression arrays as an unbiased method to investigate mechanisms underlying dasatinib-induced senescence. We performed gene expression profiling of KINSCLC cells (H1666 and Cal12T, which harbor G466VNSCLC cells (A549, H661) that were incubated for 72 hours with 150nM dasatinib or vehicle control. We selected 72 hours because we previously showed that incubation for 72 hours was required to induce irreversible senescence [3]. Using the Affymetrix Human Genome U133 Plus 2.0 array platform, we identified profiles that were modulated by dasatinib in each of the sensitive (KIH1666 and Cal12T cells) and resistant (WTA549 and H661) groups. These profiles were then cross compared among the two cell line groups to determine differential effects of dasatinib in KIrelative to WTcells (Physique ?(Figure1A).1A). We found 2061 gene features corresponding to 1458 genes that were differentially modulated by dasatinib between KIand WTcells (fold switch 1.35, 0.05; Physique ?Physique1A1A). Open in a separate window Physique 1 Comparison of gene.Singh NP, McCoy MT, Tice RR, Schneider EL. better tolerated than dasatinib, could be used to treat KINSCLC. Our novel finding that dasatinib induced DNA damage and subsequently activated DNA repair pathways leading to senescence in KINSCLC cells represents a unique vulnerability with potential clinical applications. mutations, rearrangements, or translocations. However, only a minority of the remaining 80% of patients likely have targetable, activating kinase mutations or translocations, and there is a great need to identify additional effective therapies [1]. We BLR1 previously recognized a patient with stage IV NSCLC harboring a novel mutation (Y472C) that experienced a near total radiographic response to the multitargeted kinase inhibitor dasatinib as the sole therapy; the patient lived without active malignancy for Narciclasine 7 years following treatment [2]. We discovered that Y472Cis usually a kinase-inactivating mutation (KIundergo senescence when exposed to dasatinib, whereas NSCLC with wild-type (WTand in patients [3]. The RAS/RAF/MEK/ERK pathway plays an important role in the progression of many human cancers. Once activated by surface receptors, RAS recruits RAF, a serine/threonine kinase, to the cell membrane and activates it. RAF then phosphorylates MEK, which in turn phosphorylates and activates ERK, leading to cancer progression or senescence depending on the degree of ERK activation and crosstalk with other signaling pathways [4]. The 3 RAF proteins (A, B, and C) can form homodimers and heterodimers [5]. BRAF is usually by far the most frequently mutated isoform [6]. mutations can result in increased or decreased BRAF kinase activity, as well as kinase-neutral Narciclasine mutations, and mutations occur in 3C8% of patients with NSCLC [7C11] and many other tumor types [12]. KIstill paradoxically activates MEK/ERK to levels higher than those in cells with WTvia heterodimerization with CRAF (Raf-1) [13C16]. Similarly, inhibition of WTor expression of KIincreases CRAF-BRAF binding, activates CRAF, and enhances MEK/ERK activation [3, 14C16]. The underlying mechanism of dasatinib-induced senescence in KINSCLC cells is usually obscure. Dasatinib inhibits the activity of Src and Abl, as well as nearly 40 unique kinase targets [17, 18]. Dasatinib weakly inhibits BRAF, although only at concentrations higher than those needed to induce senescence, and it can induce BRAF-CRAF dimerization and CRAF activation in cells with activated RAS or KImutations [3, 19]. Although RAF dimerization was found to be necessary for dasatinib sensitivity, nilotinib, a kinase inhibitor with a similar kinase profile that also produced strong RAF dimerization, did not induce senescence. Another potent Src/Abl inhibitor, bosutinib, did not induce senescence [3]. Currently you will find no well-defined, canonical pathways that explain the observed dasatinib-induced senescence in KINSCLC cells. We sought to define the underlying mechanism leading to dasatinib-induced senescence in KINSCLC cells. We used 2 methods: gene expression arrays and reverse phase protein array (RPPA), in which we simultaneously examined the expression of 137 proteins and phosphoproteins in KIand WTNSCLC cell lines at baseline and following dasatinib Narciclasine treatment. Our approach was limited by the existence of only 2 NSCLC cell lines with endogenous KINSCLC cells. TAZ is part of the Hippo pathway that is a complex network of at least 35 proteins that converge on a core kinase cassette that consists of MST1/2, LATS1/2, SAV1, and MOB [20]. LATS1/2 phosphorylates the transcriptional co-activators YAP and TAZ that results in their ubiquitin-mediated proteolysis. TAZ has recently been defined as a novel oncogene in NSCLC cells where TAZ knock-down results in decreased anchorage-independent growth and tumor growth and WTNSCLC cells treated with dasatinib We used gene expression arrays as an unbiased method to investigate mechanisms underlying dasatinib-induced senescence. We performed gene expression profiling of KINSCLC cells (H1666 and Cal12T, which harbor G466VNSCLC cells (A549, H661) that were incubated for 72 hours with 150nM dasatinib or vehicle control. We chose 72 hours because we previously showed that incubation for 72 hours was required to induce irreversible senescence [3]. Using the Affymetrix Human Genome U133 Plus 2.0 array platform, we identified profiles that were modulated by dasatinib in each of the sensitive (KIH1666 and Cal12T cells) and resistant (WTA549 and H661) groups. These profiles were then cross compared among the two cell line groups to determine differential effects of dasatinib in KIrelative to WTcells (Figure ?(Figure1A).1A). We found 2061 gene features corresponding to 1458 genes that were differentially modulated by dasatinib between KIand WTcells (fold change 1.35, 0.05; Figure ?Figure1A1A). Open in a separate window Figure 1 Comparison of gene expression changes following treatment.Zhou Z, Hao Y, Liu N, Raptis L, Tsao MS, Yang X. DNA damage-induced senescence. Dasatinib also led to a marked decrease in TAZ but not YAP protein levels. Overexpression of TAZ inhibited dasatinib-induced senescence. To investigate other vulnerabilities in KINSCLC cells, we compared the sensitivity of these cells with that of WTNSCLC cells to 79 drugs and identified a pattern of sensitivity to EGFR and MEK inhibitors in the KIcells. Clinically approved EGFR and MEK inhibitors, which are better tolerated than dasatinib, could be used to treat KINSCLC. Our novel finding that dasatinib induced DNA damage and subsequently activated DNA repair pathways leading to senescence in KINSCLC cells represents a unique vulnerability with potential clinical applications. mutations, rearrangements, or translocations. However, only a minority of the remaining 80% of patients likely have targetable, activating kinase mutations or translocations, and there is a great need to identify additional effective therapies [1]. We previously identified a patient with stage IV NSCLC harboring a novel mutation (Y472C) that had a near complete radiographic response to the multitargeted kinase inhibitor dasatinib as the sole therapy; the patient lived without active cancer for 7 years following treatment [2]. We discovered that Y472Cis a kinase-inactivating mutation (KIundergo senescence when exposed to dasatinib, whereas NSCLC with wild-type (WTand in patients [3]. The RAS/RAF/MEK/ERK pathway plays an important role in the progression of many human cancers. Once activated by surface receptors, RAS recruits RAF, a serine/threonine kinase, to the cell membrane and activates it. RAF then phosphorylates MEK, which in turn phosphorylates and activates ERK, leading to cancer progression or senescence depending on the degree of ERK activation and crosstalk with other signaling pathways [4]. The 3 RAF proteins (A, B, and C) can form homodimers and heterodimers [5]. BRAF is by far the most frequently mutated isoform [6]. mutations can result in increased or decreased BRAF kinase activity, as well as kinase-neutral mutations, and mutations occur in 3C8% of patients with NSCLC [7C11] and many other tumor types [12]. KIstill paradoxically activates MEK/ERK to levels higher than those in cells with WTvia heterodimerization with CRAF (Raf-1) [13C16]. Similarly, inhibition of WTor expression of KIincreases CRAF-BRAF binding, activates CRAF, and enhances MEK/ERK activation [3, 14C16]. The underlying mechanism of dasatinib-induced senescence in KINSCLC cells is obscure. Dasatinib inhibits the activity of Src and Abl, as well as nearly 40 distinct kinase targets [17, 18]. Dasatinib weakly inhibits BRAF, although only at concentrations higher than those needed to induce senescence, and it can induce BRAF-CRAF dimerization and CRAF activation in cells with activated RAS or KImutations [3, 19]. Although RAF dimerization was found to be necessary for dasatinib sensitivity, nilotinib, a kinase inhibitor with a similar kinase profile that also produced robust RAF dimerization, did not induce senescence. Another potent Src/Abl inhibitor, bosutinib, did not induce senescence [3]. Currently there are no well-defined, canonical pathways that explain the observed dasatinib-induced senescence in KINSCLC cells. We sought to define the underlying mechanism leading to dasatinib-induced senescence in KINSCLC cells. We used 2 approaches: gene expression arrays and reverse phase protein array (RPPA), in which we simultaneously examined the expression of 137 proteins and phosphoproteins in KIand WTNSCLC cell lines at baseline and following dasatinib treatment. Our approach was limited by the existence of only 2 NSCLC cell lines with endogenous KINSCLC cells. TAZ is part of the Hippo pathway that is a complex network of at least 35 proteins that converge on a core kinase cassette that consists of MST1/2, LATS1/2, SAV1, and MOB [20]. LATS1/2 phosphorylates the transcriptional co-activators YAP and TAZ that results in their ubiquitin-mediated proteolysis. TAZ has recently been defined as a novel oncogene in NSCLC cells where TAZ knock-down results in decreased anchorage-independent growth and tumor growth and WTNSCLC cells treated with dasatinib We used gene expression arrays as an unbiased method to investigate mechanisms underlying dasatinib-induced senescence. We performed gene expression profiling of KINSCLC cells (H1666 and Cal12T, which harbor G466VNSCLC cells (A549, H661) that were incubated for 72 hours with 150nM dasatinib or vehicle control. We chose 72 hours because we previously showed that incubation for 72 hours was required to induce irreversible senescence [3]. Using the Affymetrix Human Genome U133 Plus 2.0 array platform, we identified profiles that.