Comprehensive targeting of resistance to inhibition of RTK signaling pathways by using glucocorticoids

Inhibition of RTK pathways in cancer triggers an adaptive response that promotes therapeutic resistance. Because the adaptive response is multifaceted, the optimal approach to blunting it remains undetermined. TNF upregulation is a biologically significant response to EGFR inhibition in NSCLC. Here, we compared a specific TNF inhibitor (etanercept) to thalidomide and prednisone, two drugs that block TNF and also other inflammatory pathways. Prednisone is significantly more effective in suppressing EGFR inhibition-induced inflammatory signals. Remarkably, prednisone induces a shutdown of bypass RTK signaling and inhibits key resistance signals such as STAT3, YAP and TNF-NF-κB. Combined with EGFR inhibition, prednisone is significantly superior to etanercept or thalidomide in durably suppressing tumor growth in multiple mouse models, indicating that a broad suppression of adaptive signals is more effective than blocking a single component. We identify prednisone as a drug that can effectively inhibit adaptive resistance with acceptable toxicity in NSCLC and other cancers.

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A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.
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Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy RNA-seq data that support the findings of this study was deposited in the Sequence Read Archive (PRJNA763241, https://www.ncbi.nlm.nih.gov/bioproject/ PRJNA763241). Source data are provided with this paper.

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Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Sample sizes were chosen based on our previous publications, literatures, and power analysis.
Animal experiments on nude mice or NOD-SCID mice, n=8 per group, was based on our previous publications, literatures and power analysis. The power analysis parameters: 1. effect size of 1.67 was assumed on 50% tumor size reduction after 32 days treatment between two groups in comparison and a standard deviation of 30% for tumor volume in each of the comparison groups; 2. 85% power and 5% type I error; 3. twosample two-tailed t-test for two independent means.
Transgenic mouse model , n=4 in groups with TKI, was base on our previous publications, literatures and power analysis. The power analysis parameters: 1.an approximate average effect size of 2 was observed from nude mice (xenograft) and NOD-SCID mice (PDX); 2. 80% power and 5% type I error; 3. two-sample one-tailed t-test for two independent means.
Before vs After TKI treatment: The sample size of 10 (before) plus 13 (after) was based on our previous publications. Moreover, 10 is the number of all available TKI-treated NSCLC samples from Jackson(5) or UTSW(5) collected for this study, as the rarity of EGFR mutant and recurrent tumors for re-biopsy.
The power calculation on survival analysis above was performed on http://www.sample-size.net/sample-size-survival-analysis/ . Other power calculation above was calculated by GPower 3.1 software.
Data exclusions No collected data were excluded.

Replication
For cell viability, qPCR, and luciferase experiments, the experiment was done in triplicate (3 technical replicates). Data show representative of 3 independent repeats with similar results. For ELISA assay, Three independent experiments were performed. Western blot images, tumor images, MRI images, and Immunofluorescence images are representative of three independent experiments(western blot and Immunofluorescence), or indicated number of mouse tumors (tumor images, and MRI images) RNA-seq data contains 8 conditions, each has three biological independent samples. All replications above have similar results and are reproducible. Transcription factor array and Phospho-RTK Array were performed according to the protocol of these commercial kits. No repeated experiments at same conditions were performed. However, experiments were performed on multiple cell lines.
Randomization For in vivo experiments, the mice were randomly divided into control and different treatment groups, by following steps: All female 4-6 weeks old nude mice or NOD-SCID mice in the same experiment were ordered, received, injected/implanted, grouped, and treated together. After tumor formation, for randomization, all mice in one experiments would be mix together (all female) and then assigned into different groups, randomly selecting one by one, regardless of tumor sizes, in the sequences like ABBAABBA...(two groups) or ABCDDCBA...(four groups) for the purpose of balancing slow and fast running mice, as fast running mice tend to have a slow growing tumors and hard to grab.
Therefore, the groups in ABCDDCBA... form were well balanced, and allocation of control and different experiment groups can be random.
As the transgenic mice included both sexes, the male(s) could not be merged with other male(s) if not from the same colony since birth, and males could not mixed with females in one cage. Thus, the randomization should be adjusted by those 2 rules above, and each group would contain both male(s) and female(s) after randomly grouping.

Blinding
Animal data: The treatment or control groups were labelled on cage cards. The tumor size measurement every 4 days was set at another time point than treatment. While measuring, the cage cards would be flipped over at first, and length and width of tumor, as well as ear tag ID of

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each mouse would be recorded. After an experiment was done and when mice were sacrificed, the mapping from ear tag ID to group ID could be revealed.
MRI: MRI images were analyzed by ImageJ to measure tumor sizes. Mice were labelled by toe and ear clipping. The grouping and drug treatment was performed by one person(KG). The tumor MRI images collection and analysis were performed by 2 persons(KG and GG) without knowing the treatment conditions. The tumor size measurement based on MRI images was performed after all MRI images were collected and mice were sacrificed.

Mycoplasma contamination
Cells were tested negative for mycoplasma contamination using an e-Myco kit (Boca Scientific).

Commonly misidentified lines (See ICLAC register)
The study did not involve commonly misidentified lines.

Wild animals
The study did not involve wild animals.
Field-collected samples The study did not involve field-collected samples.

Ethics oversight
All animal studies were done under IACUC-approved protocols at UT Southwestern and North Texas VA Medical Center (Dallas, Texas, USA).
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants