Rosangela Aparecida Moreira Wailemann

The Y1 mouse adrenocortical tumor cell line is a classic model for cancer studies and ACTH-mediated steroidogenesis. The KRAS gene amplification and SOS-mediated activation sustain elevated ERK levels, which drive the tumor phenotype of this cell line. In addition, the mitogen FGF2 induces atypical cytotoxic stress and consequent cell-death rather than proliferation in this system, imposing a strong perturbation on KRAS-ERK signaling dynamics. Recent research from our group revealed that SOS is not solely responsible for KRAS activation in the Y1 cells, indicating a more complex regulatory landscape of GEFs and GAPs. This motivated the generation of a comprehensive time-resolved proteome (DIA) and TMT-phosphoproteome (DDA) in a 0 to 30-minute time course under FGF2 stimulation or combined with SOS inhibitor, with the presence or absence of FBS stimulation, in technical triplicate using Q-Exactive™ and Thermo Lumos™ mass spectrometers, respectively. This dataset can enable latent mechanisms between the life and death switch caused by FGF2 signaling and the alternate stress response caused by impaired SOS-dependent activation. This analysis yielded 14,512 phosphosites and 8,919 protein groups, where 10,471 phosphosites and 111 protein groups showed differential abundance with statistical significance (limma, FDR < 0.05%). This dataset provides a versatile resource for downstream applications, which includes dynamic modeling such as ordinary differential equation–based frameworks, for which temporal datasets remain scarce. The data contained in this preprint will be available on JPOST Repo after the publication.

Supplementary table 7: Full list of genes whose knockout was selectively toxic in the presence of LB-100 in SW-480 cells in the CRISPR-KO screen FDR smaller or equal to 0.25 and log2 fold change smaller or equal to -1 in treated/untreated comparison were criteria for hit selection.

Supplementary table 4: The composition of the stress-focused drug library Compounds comprising the stress-focused drug library with their respective targets.

Figure S3: High N-Myc levels sensitize neuroblastoma cells to LB-100, and PP2A knockdown sensitizes CRC cells to WEE1 inhibition (A) Western blots comparing N-Myc levels in isogenic neuroblastoma models. GAPDH was used as a loading control. (B) long-term viability assays compares the toxicity of LB-100 across these neuroblastoma models. LB-100 was refreshed every 2-3 days, and the cells were grown for 10 days before fixing, staining, and imaging. (C) Western blots show the knockdown of PPP2R1A in HT-29 and SW-480 cells. GAPDH was used as a loading control. (D) Dose-response assays show the effect of Adavosertib after PPP2R1A knockdown compared to a control shRNA in HT-29 and SW-480 cells. Cell viability was estimated by resazurin fluorescence after 5 days in the presence of the drug or DMSO control. The normalized values are plotted. (E) The heat map shows all “Hallmarks” and “KEGG” molecular signatures significantly enriched by LB-100 (4 µM), calyculin A (5 nM), or okadaic acid (10 nM) in HT-29 and SW-480 cells after 8 hours. Asterisks indicate significance level (* p-value <0.05, ** p-value <0.01, *** p-value <0.001).

Figure S7: Normal tissues from the orthotopic CRC PDXs are not affected by LB-100, adavosertib, or the combination. Representative Hematoxylin & Eosin (H&E) stainings of the heart, liver, lung, and spleen from the PDOX1 PDXs treated as indicated. Original magnifications are indicated.

Figure S9: Single-cell RNAseq identify transcriptional signatures downregulated in CRC cells after acquired resistance to the combination of LB-100 and adavosertib UMAP representations of HT-29 (A) and SW-480 (B) cells colored by the activity scores for the indicated pathways. UMAPs colored by sample of origin from both cell lines are present of the left for reference. The boxen plots show the pathway activity scores for parental (red) and resistant (blue) cells.

Figure S8: Acquired resistance to the combination of LB-100 and adavosertib suppressed malignant traits in CRC models (A) IncuCyte-based proliferation assays from HT-29 and SW-480 parental and resistant cells in the absence or presence of the combination (LB-100 4 µM + adavosertib 400 nM). (B) Chromosome counting and representative chromosome spreads from HT-29 and SW-480 parental and resistant cells. Nocodazole was added for 3h to block cells in mitosis. Cells were harvested by mitotic shake-off for spreading. Over 40 (HT-29 and HT-29-R) or 50 (SW-480 and SW-480-R) spreads were counted per cell line. Asterisks indicate significance level (**** p-value <0.0001) by two-tailed Mann-Whitney test. (C) Heatmaps showing the marker genes of each cluster from the scRNAseq analyses of HT-29 and SW-480 cells

Supplementary table 5: Stress-focused drug screens AUC differences in HT-29 cells Area Under the Curve (AUC) from each compound of the stress-focused drug screen in the presence or absence of LB-100. Compounds are ranked by the difference in the AUC between LB-100-treated and untreated samples.

Figure S10: Acquired resistance to the combination of LB-100 and Adavosertib suppress anchorage-independent and tumor growth in CRC models (A) Endpoint proliferation of HT-29 and SW-480 parental and resistant cells growing attached or in anchorage-independent conditions. Cells were plated in the same density on regular or cell-repellent culture plates in the absence of drugs and grown for 5-6 days. Proliferation was addressed by CellTiter-Glo 3D® and is expressed as fold change over T0. (B) Growth curves of the individual tumors from SW-480 parental and resistant cells measured 3 times per week. The dashed line indicates the 1500 mm3 ethical sacrifice.

Figure S1: LB-100 engages stress-related, inflammatory response, and mitogenic signaling transcriptional programs in CRC cells (A) Dose-response assays show the effect of LB-100 in 7 CRC models. Cell viability was estimated by resazurin fluorescence after 5 days in the presence of the drug or DMSO control. The normalized values are plotted. (B) The heat map shows all “Hallmarks” and “KEGG” molecular signatures significantly enriched by LB-100 (4 µM) in both HT-29 and SW-480 cells in at least one of the addressed time points. Asterisks indicate significance level (* p-value <0.05, ** p-value <0.01, *** p-value <0.001)