Figures & data: A novel lead compound CM-118

来自 : 发布时间：2024-12-25

Antitumor activity and new insight into the molecular mechanism and combination therapy strategy in c-Met- and ALK-dependent cancersFigures dataA novel lead compound CM-118Antitumor activity and new insight into the molecular mechanism and combination therapy strategy in c-Met- and ALK-dependent cancersAll authorsLanfang Meng, Mengjun Shu, Yaqing Chen, Dexiao Yang, Qun He, Hui Zhao, Zhiyong Feng, Chris Liang Ker Yuhttps://doi.org/10.4161/cbt.28409Published online:11 March 2014

Figure 1. CM-118 is a potent and selective dual inhibitor of c-Met and ALK. (A) CM-118 chemical structure is shown. (B) c-Met, ALK enzyme assays via HTRF; dose curves and mean IC50 values are shown. (C) One micromole per liter racemic mixture of CM-118 and crizotinib (PF-1066) were assayed in 96 human kinases (KINOMEscan™). Kinase inhibition dendrograms are shown; the size of red dots is proportional to the degree of inhibition. (D) Serum-starved A549 cells were stimulated by HGF with vehicle or CM-118, then immunoblotted. (E) HGF-induced A549 cell migration was assayed without or with CM-118; left, crystal violet staining of migrated cells; right, migrated cells were quantified by 5 visual fields per filter. (F) HEK293 transiently expressing the indicated ALK constructs, treated with CM-118 for 6 h then immunoblotted. The phospho-ALK levels were quantified. CM-118 IC50 (μmol/L) values are shown.

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Figure 1. CM-118 is a potent and selective dual inhibitor of c-Met and ALK. (A) CM-118 chemical structure is shown. (B) c-Met, ALK enzyme assays via HTRF; dose curves and mean IC50 values are shown. (C) One micromole per liter racemic mixture of CM-118 and crizotinib (PF-1066) were assayed in 96 human kinases (KINOMEscan™). Kinase inhibition dendrograms are shown; the size of red dots is proportional to the degree of inhibition. (D) Serum-starved A549 cells were stimulated by HGF with vehicle or CM-118, then immunoblotted. (E) HGF-induced A549 cell migration was assayed without or with CM-118; left, crystal violet staining of migrated cells; right, migrated cells were quantified by 5 visual fields per filter. (F) HEK293 transiently expressing the indicated ALK constructs, treated with CM-118 for 6 h then immunoblotted. The phospho-ALK levels were quantified. CM-118 IC50 (μmol/L) values are shown.

A novel lead compound CM-118Antitumor activity and new insight into the molecular mechanism and combination therapy strategy in c-Met- and ALK-dependent cancersAll authorsLanfang Meng, Mengjun Shu, Yaqing Chen, Dexiao Yang, Qun He, Hui Zhao, Zhiyong Feng, Chris Liang Ker Yuhttps://doi.org/10.4161/cbt.28409Published online:11 March 2014Table 1. Inhibition profile of CM-118 against a panel of ALK and ALK mutant kinases as assessed by 32p-ATP assays (Reaction Biology Corporation) with 10 μmol/L total ATPCSVDisplay TableA novel lead compound CM-118Antitumor activity and new insight into the molecular mechanism and combination therapy strategy in c-Met- and ALK-dependent cancersAll authorsLanfang Meng, Mengjun Shu, Yaqing Chen, Dexiao Yang, Qun He, Hui Zhao, Zhiyong Feng, Chris Liang Ker Yuhttps://doi.org/10.4161/cbt.28409Published online:11 March 2014

Figure 2. CM-118 inhibits proliferation of c-Met- or ALK-driven cancer cell lines. Panels of 5 NSCLC (A, left), 5 gastric (B, left), and 4 brain (C, left) cancer lines were immunoblotted as indicated. CM-118 was assessed for growth inhibition potency in corresponding cancer lines. Bar graphs show mean IC50 values (n = 3) determined from MTS assays for NSCLC (A, right), gastric (B, right), and brain (C, right) panels.

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Figure 2. CM-118 inhibits proliferation of c-Met- or ALK-driven cancer cell lines. Panels of 5 NSCLC (A, left), 5 gastric (B, left), and 4 brain (C, left) cancer lines were immunoblotted as indicated. CM-118 was assessed for growth inhibition potency in corresponding cancer lines. Bar graphs show mean IC50 values (n = 3) determined from MTS assays for NSCLC (A, right), gastric (B, right), and brain (C, right) panels.

A novel lead compound CM-118Antitumor activity and new insight into the molecular mechanism and combination therapy strategy in c-Met- and ALK-dependent cancersAll authorsLanfang Meng, Mengjun Shu, Yaqing Chen, Dexiao Yang, Qun He, Hui Zhao, Zhiyong Feng, Chris Liang Ker Yuhttps://doi.org/10.4161/cbt.28409Published online:11 March 2014Table 2. Inhibition of cancer cell proliferation by CM-118CSVDisplay TableA novel lead compound CM-118Antitumor activity and new insight into the molecular mechanism and combination therapy strategy in c-Met- and ALK-dependent cancersAll authorsLanfang Meng, Mengjun Shu, Yaqing Chen, Dexiao Yang, Qun He, Hui Zhao, Zhiyong Feng, Chris Liang Ker Yuhttps://doi.org/10.4161/cbt.28409Published online:11 March 2014

Figure 3. CM-118 inhibits oncogenic c-Met signaling and survival in c-Met-driven H1993 and SNU-5 cells. H1993 (A) or SNU-5 (B) cells were treated for 6 h with various doses of CM-118, 1 μmol/L PF2341066 (PF). Total cell lysates were immunoblotted as indicated. (C) Cell lysates of H1993 or SNU-5 after 6 h treatment with various doses of CM-118, 1 μM PF, or 1 μM AZD8055 (AZD) were immunoblotted. (D) H1993 and SUN-5 cells were treated with various doses of CM-118 for 48 h, then collected for cell cycle analysis.

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Figure 3. CM-118 inhibits oncogenic c-Met signaling and survival in c-Met-driven H1993 and SNU-5 cells. H1993 (A) or SNU-5 (B) cells were treated for 6 h with various doses of CM-118, 1 μmol/L PF2341066 (PF). Total cell lysates were immunoblotted as indicated. (C) Cell lysates of H1993 or SNU-5 after 6 h treatment with various doses of CM-118, 1 μM PF, or 1 μM AZD8055 (AZD) were immunoblotted. (D) H1993 and SUN-5 cells were treated with various doses of CM-118 for 48 h, then collected for cell cycle analysis.

A novel lead compound CM-118Antitumor activity and new insight into the molecular mechanism and combination therapy strategy in c-Met- and ALK-dependent cancersAll authorsLanfang Meng, Mengjun Shu, Yaqing Chen, Dexiao Yang, Qun He, Hui Zhao, Zhiyong Feng, Chris Liang Ker Yuhttps://doi.org/10.4161/cbt.28409Published online:11 March 2014

Figure 4. CM-118 and its combination with afatinib induce apoptosis in H1993 cells. (A) H1993 plated in 6-well culture plates were treated for 48 h with 2 μmol/L CM-118, 2 μmol/L afatinib, or a combination of the two inhibitors, then assessed for viability by trypan-blue cell counting. Percent net growth or death is relative to the initial cell density at initiation of treatment. (B) Cells as in (A) were immunoblotted. H1993 cells infected with the indicated lantivirus shRNAs were similarly treated and subjected to analysis of immunoblotting (C) and cell survival (D). H1993 cells transiently transfected with the indicated expression constructs were similarly analyzed by immunoblotting (E) and assayed for survival (F). Statistical analysis was performed on cells of Sh-Bim vs. Sh NT; cells with Mcl-1 overexpression vs. vector-transfected cells.

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Figure 4. CM-118 and its combination with afatinib induce apoptosis in H1993 cells. (A) H1993 plated in 6-well culture plates were treated for 48 h with 2 μmol/L CM-118, 2 μmol/L afatinib, or a combination of the two inhibitors, then assessed for viability by trypan-blue cell counting. Percent net growth or death is relative to the initial cell density at initiation of treatment. (B) Cells as in (A) were immunoblotted. H1993 cells infected with the indicated lantivirus shRNAs were similarly treated and subjected to analysis of immunoblotting (C) and cell survival (D). H1993 cells transiently transfected with the indicated expression constructs were similarly analyzed by immunoblotting (E) and assayed for survival (F). Statistical analysis was performed on cells of Sh-Bim vs. Sh NT; cells with Mcl-1 overexpression vs. vector-transfected cells.

A novel lead compound CM-118Antitumor activity and new insight into the molecular mechanism and combination therapy strategy in c-Met- and ALK-dependent cancersAll authorsLanfang Meng, Mengjun Shu, Yaqing Chen, Dexiao Yang, Qun He, Hui Zhao, Zhiyong Feng, Chris Liang Ker Yuhttps://doi.org/10.4161/cbt.28409Published online:11 March 2014

Figure 5. CM-118 inhibits EML4-ALK signaling, survival, and its mechanism in H2228 cells. (A) H2228 cells were treated for 6 h with various doses of CM-118, 1 μmol/L PF-02341066 (PF), then immunoblotted. (B) H2228 cells were treated with the indicated CM-118 for 48 h, subjected to cell cycle analysis (top) and immunoblotting (bottom). (C) H2228 cells were treated for 24 h with the indicated CM-118, 1 μmol/L PF, rapamycin (Rapa), or AZD8055 (AZD), then immunoblotted. (D) H2228 cells were treated with the indicated inhibitors for 24 h, then stained with JC-1 and photographed as described in Methods. (E) H2228 cells were infected with GIPZ ShRNA lentivirus encoding non-targeting (NT), mTOR (Sh#2), Raptor (Sh#2), and Rictor (Sh#4). GFP-expressing (Sh-positive) viable cells were counted on day 1 and day 8 (day 6 and 14 post infection) (top) and quantified (bottom left). Puromycin-selected cells were immunoblotted (bottom right). (F) H2228 cells were treated for 3 d with 3 μmol/L CM-118, 0.1 μmol/L rapamycin (Rapa), or 0.1 μmol/L AZD8055 (AZD), then assessed for viability via trypan-blue cell counting. Results are processed similarly as in Figure 4A.

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Figure 5. CM-118 inhibits EML4-ALK signaling, survival, and its mechanism in H2228 cells. (A) H2228 cells were treated for 6 h with various doses of CM-118, 1 μmol/L PF-02341066 (PF), then immunoblotted. (B) H2228 cells were treated with the indicated CM-118 for 48 h, subjected to cell cycle analysis (top) and immunoblotting (bottom). (C) H2228 cells were treated for 24 h with the indicated CM-118, 1 μmol/L PF, rapamycin (Rapa), or AZD8055 (AZD), then immunoblotted. (D) H2228 cells were treated with the indicated inhibitors for 24 h, then stained with JC-1 and photographed as described in Methods. (E) H2228 cells were infected with GIPZ ShRNA lentivirus encoding non-targeting (NT), mTOR (Sh#2), Raptor (Sh#2), and Rictor (Sh#4). GFP-expressing (Sh-positive) viable cells were counted on day 1 and day 8 (day 6 and 14 post infection) (top) and quantified (bottom left). Puromycin-selected cells were immunoblotted (bottom right). (F) H2228 cells were treated for 3 d with 3 μmol/L CM-118, 0.1 μmol/L rapamycin (Rapa), or 0.1 μmol/L AZD8055 (AZD), then assessed for viability via trypan-blue cell counting. Results are processed similarly as in Figure 4A.

A novel lead compound CM-118Antitumor activity and new insight into the molecular mechanism and combination therapy strategy in c-Met- and ALK-dependent cancersAll authorsLanfang Meng, Mengjun Shu, Yaqing Chen, Dexiao Yang, Qun He, Hui Zhao, Zhiyong Feng, Chris Liang Ker Yuhttps://doi.org/10.4161/cbt.28409Published online:11 March 2014

Figure 6. CM-118 shows in vivo efficacy in xenograft SNU-5, U87MG tumors. (A) Nude mice bearing SNU-5 tumors were dosed orally with vehicle, 50 or 100 mg/kg CM-118. Four hours later, levels of phosphorylated c-Met and c-Met in tumor tissue were analyzed as described in Methods. (B) Tumor bearing mice were dosed orally with vehicle, 50 or 100 mg/kg CM-118 bid (n = 13). Tumor volumes (top) and body weights (bottom) are shown. (C) SNU-5 tumor-bearing mice were treated with vehicle, 12.5 or 25 mg/kg CM-118 bid (n = 5). (D) U87MG tumor-bearing mice were treated with vehicle, 15, 30, or 60 mg/kg CM-118 bid (n = 8). Tumor volumes are shown. Statistical analysis: **P 0.01; ***P 0.001. (E) On last day of study in C, mouse plasma (n = 3) were analyzed for CM-118 concentration and 8 h exposure AUC.

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Figure 6. CM-118 shows in vivo efficacy in xenograft SNU-5, U87MG tumors. (A) Nude mice bearing SNU-5 tumors were dosed orally with vehicle, 50 or 100 mg/kg CM-118. Four hours later, levels of phosphorylated c-Met and c-Met in tumor tissue were analyzed as described in Methods. (B) Tumor bearing mice were dosed orally with vehicle, 50 or 100 mg/kg CM-118 bid (n = 13). Tumor volumes (top) and body weights (bottom) are shown. (C) SNU-5 tumor-bearing mice were treated with vehicle, 12.5 or 25 mg/kg CM-118 bid (n = 5). (D) U87MG tumor-bearing mice were treated with vehicle, 15, 30, or 60 mg/kg CM-118 bid (n = 8). Tumor volumes are shown. Statistical analysis: **P 0.01; ***P 0.001. (E) On last day of study in C, mouse plasma (n = 3) were analyzed for CM-118 concentration and 8 h exposure AUC.

A novel lead compound CM-118Antitumor activity and new insight into the molecular mechanism and combination therapy strategy in c-Met- and ALK-dependent cancersAll authorsLanfang Meng, Mengjun Shu, Yaqing Chen, Dexiao Yang, Qun He, Hui Zhao, Zhiyong Feng, Chris Liang Ker Yuhttps://doi.org/10.4161/cbt.28409Published online:11 March 2014

Figure 7. CM-118 shows in vivo efficacy in c-Met-, EML4-ALK-driven NSCLC models. (A) Mice bearing the H1993 tumors (n = 8) were dosed orally with vehicle, 15, 30, 60 mg/kg bid, or 50 mg/kg PF-02341066 (PF) qd for 25 d. Tumor volumes (top) and body weights (bottom) are shown. (B) Tumor lysates made 4 h post the last dose were immunoblotted (top) and quantified (bottom). (C) Mice bearing the H2228 tumors (n = 3) were dosed once with 15 or 30 mg/kg CM-118. Tumor lysates made 4 h post dosing were immunoblotted (left) and quantified (right). (D) Plasma samples (n = 3) of mice received 15 mg/kg CM-118 were analyzed for drug concentration and 8 h exposure AUC. (E) Mice bearing the H2228 tumors (n = 8) were dosed orally with vehicle, 15, 30, 60 mg/kg bid or 50 mg PF qd for 28 d. Tumor volumes are shown. Statistical analysis for (A and E): ***P 0.001; treated vs. vehicle control. (F) Relative tumor volume (RTV) on day 28 for study groups (n = 8) of vehicle control, 15 mg/kg CM-118 bid, 10 mg/kg rapamycin qw, and combination of CM-118 and rapamycin. Statistical analysis: *P 0.05; **P 0.01.

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Figure 7. CM-118 shows in vivo efficacy in c-Met-, EML4-ALK-driven NSCLC models. (A) Mice bearing the H1993 tumors (n = 8) were dosed orally with vehicle, 15, 30, 60 mg/kg bid, or 50 mg/kg PF-02341066 (PF) qd for 25 d. Tumor volumes (top) and body weights (bottom) are shown. (B) Tumor lysates made 4 h post the last dose were immunoblotted (top) and quantified (bottom). (C) Mice bearing the H2228 tumors (n = 3) were dosed once with 15 or 30 mg/kg CM-118. Tumor lysates made 4 h post dosing were immunoblotted (left) and quantified (right). (D) Plasma samples (n = 3) of mice received 15 mg/kg CM-118 were analyzed for drug concentration and 8 h exposure AUC. (E) Mice bearing the H2228 tumors (n = 8) were dosed orally with vehicle, 15, 30, 60 mg/kg bid or 50 mg PF qd for 28 d. Tumor volumes are shown. Statistical analysis for (A and E): ***P 0.001; treated vs. vehicle control. (F) Relative tumor volume (RTV) on day 28 for study groups (n = 8) of vehicle control, 15 mg/kg CM-118 bid, 10 mg/kg rapamycin qw, and combination of CM-118 and rapamycin. Statistical analysis: *P 0.05; **P 0.01.

Table 1. Inhibition profile of CM-118 against a panel of ALK and ALK mutant kinases as assessed by 32p-ATP assays (Reaction Biology Corporation) with 10 μmol/L total ATPKinasesCM-118 IC50 (μmol/L)ALK0.007ALK (C1156Y)0.012ALK (G1202R)0.054ALK (F1174L)0.012ALK (L1196M)0.050ALK (R1275Q)0.014

CM-118 was tested in a 10-dose IC50 mode with 3-fold serial dilution starting at 10 μmol/L. IC50 values are shown.

Table 2. Inhibition of cancer cell proliferation by CM-118 TumorCell lineIC50 (µmol/L)1NSCLCH197517.41 ± 1.292A54916.42 ± 1.593HCC8279.03 ± 1.614H22281.16 ± 0.435H19930.54 ± 0.066Gastric/esophagealBGC-82320.327AGS14.078TE-119.199Eca-10918.3610MGC80–325.8211HGC-2713.14 ± 312SGC-790160 ± 813NCI-N8750.54 ± 714SNU-16.7 ± 0.315SNU-50.17 ± 0.0816BrainSH-SY5Y2.33 ± 0.6517U-87MG6.59 ± 1.3118LN-22931.90 ± 0.6319LN-1835.06 ± 4.0520LiverRBE4.79 ± 0.9321BEL-740220.89 ± 1.1322BEL-740417.65 ± 1.5323SMMC-772117.65 ± 1.5324HepG2≈6025QGY770132.01 ± 4.5826QGY770319.93 ± 1.0027HuH-77.65 ± 0.9928SK-HEP-122.82 ± 2.4029RenalCaki21.51 ± 1.0930A49822.88 ± 0.6231786-O22.31 ± 0.3232PancreaticCapan-119.35 ± 5.7933ColonHT2919.27 ± 1.4934HCT11611.36 ± 3.02

The indicated tumor cells were plated in 96-well culture plates for 24 h, treated for 72 h with DMSO or various concentrations (0.027 to 60 μmol/L) of CM-118, n ≥ 3. Cell growth was measured by MTS assays. Dose response curves were generated for determination of IC50 for each cell line.

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