Audrey Lafargue

Supplementary Figure S1 shows tumor volumes in LX33 PDX in response to control, citalopram, chemoradiation, and chemoradiation+citalopram treatments

Small cell lung cancer (SCLC) is characterized by early metastasis, intrinsic chemoradiation resistance, and tumor recurrence. Besides the lack of potentially targetable oncogenic drivers, therapeutic advancements are also hindered by the scarcity of surgically resected tissue specimens ideal for profiling studies. We used patient-derived xenografts (PDX) to model SCLC chemoradiation resistance and identified chemoradiation resistance candidate genes using RNA sequencing. Additionally, we used human SCLC cell lines to confirm our in vivo results and delineate the underlying mechanism. Transcriptome profiling showed that the Traf2- and Nck-interacting kinase (TNIK) gene was consistently upregulated in an array of SCLC PDXs exposed to chemoradiation compared with monotherapy, which is consistent with previous observations of TNIK amplification in human samples. Genetic depletion (P < 0.01) or pharmacologic inhibition (P < 0.0001) of TNIK reduced in vitro clonogenic survival of TNIKhigh SCLC cells and promoted sensitivity to chemoradiation. In vivo, pharmacologic inhibition of TNIK enhanced chemoradiation sensitivity (P < 0.0001) of the H446 cell line–derived xenograft (CDX) in NOD-SCID mice. Furthermore, pharmacologic inhibition of TNIK in vivo demonstrated sensitivity (P < 0.0001) to chemoradiotherapy (CRT) in LX33 PDX. These results indicate that TNIK plays a role in conferring resistance to chemoradiation in SCLC cell lines and in vivo in SCLC CDX and PDX models. Delineating the mechanism behind radiosensitization suggested that TNIK inhibition may impair the DNA damage response in irradiated cells. Collectively, these findings suggest that TNIK may be a promising therapeutic target in limited-stage SCLC and support further investigation of TNIK inhibition in combination with standard CRT.

Supplementary Figure S3 shows: a) Volcano plots of differentially expressed genes in chemoradiation-treated versus control conditions across five individual PDX models. b) Heatmap displaying the enrichment scores for WikiPathways gene sets with significant enrichment in at least 3 PDXs.

Supplementary Figure S2 shows a scatter plot showing TNIK expression versus normalized tumor volume in both control and chemoradiotherapy groups across 5 PDX models

Supplementary Figure S4 shows long-term cell viability by clonogenic assay showing that TNIK-high SCLC cells (H446) are more sensitive to NCB-0846 (300nM) than TNIK-low SCLC cells (DMS114).

Supplementary Figure S5 shows: a) Response to treatment with either cisplatin or etoposide for 48h in SCLC cell lines, determined by the CellTiter-Glo 2.0 assay. b) Synergy assessment of NCB-0846 in combination with cisplatin or etoposide in H446 and DMS114 cells using the zero-interaction potency model (ZIP).

Supplementary Figure S6 shows clonogenic survival of TNIK-high H446 and TNIK-low DMS114 cells treated with vehicle, 0.5 uM cisplatin, 300 nM NCB-0846, or both for 48h before receiving radiation.

Supplementary Figure S8 shows the activation status of histone H3 in (A) TNIK-high H446 cells pre-treated with ethanol vs. NCB-0846 (300 nM, 1 and 24h) post-IR (6 Gy) and in (B) TNIK-overexpressing and mCherry-overexpressing (control) DMS114 cells post-IR (6 Gy), as determined by immunoblotting.

Supplementary Figure S7 shows the activation status of DDR proteins (yH2AX, ATM/p-ATM, Chk2/p-Chk2) in TNIK-overexpressing and mCherry-overexpressing (control) DMS114 cells post radiation (6 Gy), as determined by immunoblotting.

Supplementary Figure S9 shows the frequency of mitotic catastrophe in (A) H446 cells transfected with scramble control vs. TNIK siRNA (75 nM, 48h) post-IR (6 Gy) and in (B) TNIK-overexpressing vs mCherry-overexpressing (control) DMS114 cells post-IR (6 Gy).