Sns 032

CYC202 (seliciclib, R-Roscovitine), CCT68127, and CYC065 were provided by Cyclacel Ltd. Cycloheximide (C4859) and ActD (A9415) were purchased from MilliporeSigma and MG132 (1748) from Tocris Bioscience. Temozolomide, flavopiridol, palbociclib, dinaciclib, and SNS-032 were purchased from SelleckChem. BAY 1145372 was purchased from Active Biochem. Compound 3 was provided by Keith Jones (ICR). THZ1 (A8882) was purchased from Stratech. NVP-2 was obtained from Calla Olson, Baylor College of Medicine. THAL-SNS-032 was synthesized in house (27 (link)).

Cancer cells or HBECs were plated at 8000 cells per well in 96-well plates and cultured for overnight, then were treated with agents for 24 h to examine agent cytotoxicity. The tested agents include MSC-EVs, MSCTRAIL-EVs (MSCT-EVs), recombinant TRAIL (rTRAIL; amino acids 114–281, Peprotech), the pan-caspase inhibitor Z-VAD-FMK (1 mg/ml, Sigma), a neutralising monoclonal anti-TRAIL antibody (10 ng/ml, Sigma, Cat. No. T3067), a CDK9 selective inhibitor SNS032 (S1145, Selleckchem, UK) (300 nmol/l) and control medium, which were added to culture medium in combination or alone. After treatment, both floating and adherent cells were collected and stained with AF647-conjugated Annexin V (Invitrogen) and 2 μg/ml DAPI (Sigma), followed by cell death assessment by means of flow cytometry. Annexin V+/DAPI− cells were considered to have undergone early apoptosis, Annexin V+/DAPI+ staining considered as late apoptosis, Annexin V−/DAPI+ cells considered to be dead not by apoptosis and both Annexin V and DAPI negative cells were regarded as viable. In parallel, cell apoptosis was also assessed using the Annexin V-PE/7-AAD cell apoptosis assay kit (Cat: 559763, BD-Pharmingen) following the manufacturer’s instructions.

The following small molecule compounds targeting the RTK/Ras/PI3K pathway were diluted in DMSO (Sigma-Aldrich, St. Louis, USA): lapatinib ditosylate was obtained from Santa Cruz Biotechnology (Dallas, USA) (catalog number 202205A). Crizotinib (S1068), NVP-BKM120 (S2247), GDC-0068 (S2808), AZD2014 (S2783), AZD8055 (S1555) and GSK2636771 (S8002) were obtained from Selleck Chemicals (Houston, USA).
The following small molecule compounds targeting the Rb pathway were diluted in DMSO: SNS-032 (S1145) was obtained from Selleck Chemicals. Palbociclib isethionate (PD-0332991) (S1579) was diluted in milliQ water and was obtained from Selleck Chemicals.
The following small molecule compounds targeting the p53 pathway were diluted in DMSO: Nutlin3 (S1061) and Alisertib (MLN8237) (S1133) were obtained from Selleck Chemicals. PRIMA-1MET (SC-361295) was obtained from Santa Cruz Biotechnology.
The following small molecule compound was identified as a potential combination partner of AZD8055 and was diluted in DMSO: ABT-263 (11500) was obtained from Cayman Chemical (Ann Arbor, USA).

A kinase inhibitor library, collection of 193 kinase inhibitors, and identified specific kinase inhibitors (HMN-214, AT7519, SNS-032, and KX2-391) were purchased from Selleckchem (Houston, TX, USA). All chemicals were dissolved in dimethyl sulfoxide (DMSO) before treatment, and the cells were treated with various concentrations. To screen small-molecule compounds that modulate luc-21-miRDREL A549 cells (1 × 10⁴ cells/well) in a 96-well plate were treated with 0.1, 0.2, 1, and 4 μM of kinase inhibitor library for 48 h. Cells were assayed with a Dual-Luciferase Reporter Assay System (Promega).

LDC526 [(3-((4-(4-fluoro-2-methoxyphenyl)-1,3,5-triazin-2-yl)amino)phenyl)methane-sulfonamide, molecular weight 389,41 g/mol, Figure 1A] was provided as powder (LDC526 synthesis, supplementary methods). For oral gavage administration LDC526 was diluted in polyethelene glycol (PEG M_n 400, Sigma-Aldrich, Munich, Germany) followed by sonication for 3 x 5 minutes with intermediate mixing. The CDK inhibitors SNS-032, R-547 (Selleckchem, Munich, Germany) and Flavopiridol (Sigma-Aldrich) were diluted in DMSO.