Alex W. Tan

Supplementary Materials and Methods

Fig. S2 Venn diagram of the number of significant siRNAs identified with a 5% false discovery rate using different normalization methods (Z-score, b-score, and regional scaling) for comparison between untreated and treated samples.

Fig. S4 Effect of combination of drugs on SUM149-R cells. The clonogenic assay shows a significant dose-dependent decrease in the colony formation capability of SUM149-R cells after treatment with the combination of MEKK inhibitor AZD6244 and MCL-1 inhibitor S63845 at the same time.

Table S2 List of top 26 targets identified in the whole-genome siRNA screening.

Table S1 Quantitative PCR primers used in the study.

Fig. S3 CTB assay showing the effect of combination of drugs on SUM149-R cells. The SUM149-R cells showed a decrease in viability after treatment with the constant doses of AZD5991 (0.5, and 1µM) followed by the different doses of AZD6244.

Fig. S1 Comparative analysis of expression of MDR genes in the parental and resistant cells.

The MAPK pathway can drive resistance in highly aggressive breast cancers. Our previous work showed that the MEK inhibitor (MEKi) AZD6244 (selumetinib) prevented lung metastasis in a breast cancer xenograft model. In clinical studies, MEKis as single agents have had only modest activity against solid tumors due to the onset of resistance. Using synthetic lethality siRNA screening, we identified myeloid cell leukemia-1 (MCL-1) as a potential contributor to AZD6244 resistance. We hypothesized that MCL-1 promotes MEKi resistance in highly aggressive breast cancers and that MCL-1 inhibition overcomes AZD6244 resistance. We established two AZD6244-resistant cell lines: MDA-MB-231-R (triple-negative breast cancer) and SUM149-R (triple-negative inflammatory breast cancer). These resistant cells were characterized with respect to different parameters, and a combination of an MCL-1 inhibitor (MCL-1i) together with an MEKi was evaluated in vitro and in vivo to overcome the acquired resistance. Compared with their respective parental cells, MDA-MB-231-R and SUM149-R cells showed increased proliferation, colony formation, stemness, anchorage-independent growth, and MCL-1 expression levels. MCL-1 knockdown in resistant cells decreased cell proliferation and colony formation, increased apoptosis, and was associated with high expression of the proapoptotic proteins PUMA, NOXA, BAK, and BAX. MEKi resistance was overcome when resistant cells were treated with MCL-1i and MEKi combined. In an in vivo mouse model, inhibition of MCL-1 restored sensitivity to AZD6244. Our results suggest that MCL-1 is a driver of MEKi resistance and that combining an MCL-1i with an MEKi warrants further investigation in triple-negative and triple-negative inflammatory breast cancer.

Supplementary Figure S3. Top 20 master regulators that were differentially expressed in Cas9-p15 control and MELK knockout (KO) MDA-NB-231 cells.

Supplementary Materials and Methods