Abstract Background: Inflammatory breast cancer (IBC) is an aggressive form of primary breast cancer marked by rapid progression and a high propensity for metastasis. Despite its severity, no specific FDA-approved targeted therapies exist for IBC, highlighting the need for novel therapeutic strategies. Through analysis of metastatic xenograft-derived IBC sublines, we identified soluble E-cadherin (sEcad), a proteolytic extracellular fragment of full-length E-cadherin, as a protein associated with tumor aggressiveness and metastasis. Mass spectrometry-based proteomics further identified Protein Disulfide Isomerase A4 (PDIA4) as a novel sEcad-binding partner. We hypothesize that sEcad promotes IBC tumor progression via PDIA4-dependent mechanisms. Methods: sEcad was stably overexpresed in MDA-IBC3 and SUM149 IBC cells via lentiviral transduction, and PDIA4 was silenced using lentiviral shRNAs. Modified cells were injected into cleared mammary fat pads of SCID/Beige mice to assess tumor growth and progression. Protein-protein interactions were validated by co-immunoprecipitation. RNA-seq and GSEA were used to identify enriched pathways. Serum sEcad levels from IBC patients (n=301) and healthy donors (n=20) were measured by ELISA. Results: Serum sEcad levels were significantly higher in IBC patients than in healthy donors (P<0.0001). Elevated sEcad levels were associated with metastatic disease (p=0.0036), reduced overall survival (p=0.04), and increased risk of metastasis (0.004). Functionally, sEcad overexpression enhanced cell survival and colony formation in vitro, and mice injected with sEcad-overexpressing SUM149 or MDA-IBC3 cells exhibited accelerated tumor growth compared with controls (SUM149: p=0.007; MDA-IBC3: p=0.006). Mechanistically, endogenous and exogenous co-immunoprecipitation confirmed the interaction between sEcad and PDIA4. RNA-seq and GSEA revealed significant enrichment of NF-κB signaling in sEcad high cells. sEcad overexpression increased PDIA4 expression and NF-kB activation, whereas PDIA4 knockdown suppressed NF-κB activation and increased cell death. PDIA4 overexpression also enhanced NF-κB activation in IBC cells. In vivo, PDIA4 knockdown markedly reduced tumor incidence and tumor burden in sEcad-high IBC mouse models. Conclusions: Our study identifies sEcad functions as a clinically relevant biomarker and a driver of IBC progression via activation of PDIA4-dependent NF-kB signaling. These findings support sEcad and PDIA4 as mechanistically linked contributors to IBC and highlight this pathway as a potential therapeutic target. Citation Format: Xiaoding Hu, Kiros Haddish Tesfamariam, Isabella L. Rizzo, Emilly S. Villodre, Lan Thi Hanh Phi, Juhee Song, Yun Gong, Savitri Krishnamurthy, Wendy A. Woodward, Bisrat G. Debeb. PDIA4-NF-κB signaling mediates soluble E-cadherin-driven inflammatory breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB280.

Background:Inflammatory breast cancer (IBC) is a rare and highly aggressive form of breast cancer with an increased propensity to metastasize to distant organs including the brain. Higher serum levels of the calcium-binding proteins S100A8/A9, particularly of S100A9, have emerged as a clinically and biologically significant factor in aggressive breast cancers that are associated with poorer prognosis, tumor progression, and resistance to therapy. However, its contribution in IBC specifically remains undefined. Here, we investigated whether serum levels of S100A8/A9 predict outcomes in patients with IBC. Methods:Serum S100A8/A9 levels were measured in a cohort of 304 IBC patients using ELISA assay. S100A8/A9 levels were categorized by their third quartile value (S100A8/A9-low ≤ 3rd quartile; S100A8/A9-high > 3rd quartile). Overall survival (OS) and breast cancer-specific survival (BCSS) were analyzed with Kaplan-Meier curves, log-rank tests, and Cox proportional hazard regression models. The cumulative incidence of any metastases and the cumulative incidence of brain metastases were analyzed using Aalen-Johansen method, Gray test, and Fine-Gray models. Results:The median follow-up time was 64 months. Forty-six percent of patients had estrogen receptor (ER)-negative tumors, 61.3% were stage III-IV, 77% high grade, 16.8% received adjuvant chemotherapy and 53.6% received adjuvant radiation. On univariate analysis, S100A8/A9 levels, disease stage, ER status, PR status, HER2 status, adjuvant chemotherapy, and adjuvant radiation therapy were significantly associated with OS and BCSS. Patients with high S100A8/A9 serum levels had poor OS (P=0.01) and BCSS (P=0.007) and had a higher risk of developing brain metastasis (P=0.01) but not other metastasis. On multivariate analysis, high S100A8/A9 serum levels were independently associated with reduced OS (hazard ratio [HR]=1.7, 95% CI 1.1 to 2.6, P=0.01), reduced BCSS (HR=1.8, 95% CI 1.2 to 2.8, P=0.006), and increased cumulative incidence of developing brain metastasis (subdistribution hazard ratio (sHR)=1.8, 95% CI 1.1 to 3.0, P=0.03). Conclusions:In patients with IBC, high serum levels of S100A8/A9 are an independent prognostic factor for brain metastasis and poor clinical outcomes. These findings support the potential of S100A8/A9 as predictive biomarker for identifying increased risk of brain metastasis and unfavorable prognosis in patients with IBC.

Abstract Highly aggressive cancers such as inflammatory breast cancer (IBC) are characterized by the presence of tumor cell emboli within lymphatic and blood vessels, a phenomenon known as lymphovascular space invasion (LVSI). In this study, we developed a vascularized 3D in vitro tumor microfluidic platform to evaluate LVSI mechanisms including tumor invasion, cancer cells intravasation, vascular and Extracellular Matrix (ECM) remodeling, and emboli formation in IBC cell lines (MDA-IBC3, A3250, and SUM149).Initially, various collagen concentrations (2,3,4,5and 6 mg/mL) were evaluated using optical fiber-based interferometry nanoindentation to measure collagen hydrogel stiffness and identify a concentration representative of normal breast tissue (0.5-1 kPa). A 4 mg/mL collagen concentration was selected, yielding an average stiffness of approximately 0.7 kPa, and was used to assess how the incorporation of cancer cells alters ECM stiffness. Platforms were fabricated by polymerizing a 4 mg/mL collagen type I solution containing cancer cells (2 million cells/mL) around two 22G needles to form vascular channels. These channels were seeded with mKate-tagged telomerase-immortalized endothelial (TIME) cells at 10 million cells/mL, and a shear stress of 4 dyn/cm2 was applied to establish aligned, functional endothelium. Confocal microscopy was used to monitor vessel sprouting, permeability, emboli formation, and tumor cell intravasation for one week. Effluent media was collected on Days 3 and 7 for cytokine analysis. By day 7, vessel coverage was significantly reduced in TIME+A3250 and TIME+SUM149 platforms compared to TIME-only and TIME+IBC3 platforms (p < 0.001). Although the number of endothelial sprouts was significantly higher in TIME+A3250 platforms compared to TIME+IBC3, the average sprout length did not differ significantly. A3250 cells also exhibited a higher frequency of invadopodia-positive tumor cells compared to MDA-IBC3, indicating a greater invasive phenotype. Consistent with this, the number of intravasated A3250 cells was approximately 15-fold higher than that of MDA-IBC3 cells. Spatial analyses of the platforms’ midplanes images further revealed that A3250 cells accumulated around the vessel to a much greater extent than the area farther from the vessel over time. Notably, platforms containing A3250 cells showed an extreme hydrogel deformation, including central separation, suggesting profound collagen remodeling driven by this cell line. This trend was not observed with the other IBC lines (MDA-IBC3 and SUM149).In conclusion, our results indicate that the A3250 IBC cell line shows a more aggressive phenotype compared to the other IBC cell lines. We observed significantly higher intravasation relative to MDA-IBC3, greater disruption of endothelial vessel coverage, and a higher frequency of invadopodia-positive cells. Citation Format: Melika Mehrabi Dehdezi, Ali Moghaddaszadeh, Zoe Apsel, Surbhi Shivhare, Xiaoding Hu, Bisrat G. Debeb, Wendy A. Woodward, Marissa Nichole Rylander. A vascularized 3D microfluidic breast tumor platform for characterizing lymphovascular space invasion among inflammatory breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3409.

Abstract Background: Inflammatory breast cancer (IBC) is a rare but highly aggressive variant of breast cancer, accounting for 10% of breast cancer-related deaths. We have identified NDRG1, located on chromosome 8q24.3 near MYC within a commonly amplified region, as a key promoter of tumor growth and progression in IBC models and is associated with poor survival outcomes. Unlike many oncogenes, NDRG1 is rarely deleted or silenced and is more frequently expressed in IBC compared to non-IBC tumors. A recent study reported NDRG1 amplification in 42% of triple-negative IBC cases. We hypothesize that NDRG1 amplification in triple-negative IBC is associated with distinct gene expression profiles and enrichment of oncogenic pathways that contribute to the aggressive phenotype of the disease. Methods: RNA sequencing data from 19 triple-negative IBC tumors (8 NDRG1 amplified, 11 non-amplified) were analyzed using DEseq2 package. Heatmaps were generated to visualize clustering and expression differences. Gene Set Enrichment Analysis (GSEA) was performed using Hallmark and KEGG pathway databases. Publicly available TCGA and METABRIC breast cancer datasets were used to assess NDRG1 amplification and its association with mRNA/protein expression and survival outcomes. Results. Among the 19 triple-negative tumors analyzed, 8 (42%) exhibited NDRG1 amplification, which was significantly correlated with increased RNA expression (p = 0.05). Differential expression analysis identified CALCA, RHO, DPYSL5, GPR101, ZIC3, and IRS4 as the most upregulated, and SERPINA6, SCGB3A1, PI3, LRG1, DAPL1, MYEOV as the most downregulated in NDRG1-amplified tumors. GSEA revealed enrichment of DNA repair, cell cycle, mTOR signaling, and MYC target pathways, and downregulated pathways included interferon gamma response, estrogen response and NFkB pathway. TCGA and METABRIC analyses confirmed that NDRG1 amplification correlates with elevated mRNA expression (p<0.0001) and elevate protein levels (p<0.0001) and poorer overall survival (p=0.013, TCGA; p=0.0002, METABRIC). Conclusions: NDRG1 amplification marks a transcriptionally distinct subset of triple-negative IBC, enriched in proliferative and DNA repair pathways. These molecular features highlight its potential as a biomarker of aggressive disease biology and potential target for further mechanistic investigation and therapeutic intervention. Citation Format: Emilly S. Villodre, Ganiraju Manyam, Xiaoding Hu, Isabella L. Rizzo, Lan H. Phi, Kiros H. Tesfamariam, Azadeh Nasrazadani, Rachel M. Layman, Bora Lim, Vicente Valero, Savitri Krishnamurthy, The MDACC IBC Team, MDACC Rare Tumor Initiative Team, Jing Wang, Xiaoping Wang, Naoto Ueno, Wendy A. Woodward, Bisrat G. Debeb. Transcriptomic analysis of NDRG1 amplification in aggressive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1181.

BACKGROUND:The brain is a common site of relapse in inflammatory breast cancer (IBC), an E-cadherin-positive, aggressive form of breast cancer. Elevated serum levels of soluble E-cadherin (sEcad), an 80-kDa fragment, correlated with poorer outcomes and increased brain metastases in patients with metastatic IBC. We hypothesize that sEcad is a driver of brain metastasis in IBC. METHODS:Serum sEcad levels from 348 IBC patients were quantified by ELISA. To examine sEcad function, we used recombinant sEcad protein and generated stable IBC cell lines by cloning and overexpressing Flag-tagged sEcad. Control and sEcad-overexpressing MDA-IBC3 and SUM149 cells were injected into SCID/Beige mice to evaluate brain metastasis burden and survival, and a brain-permeable CXCR2 inhibitor was also tested for efficacy in these models. RESULTS:Higher serum sEcad levels correlated with poorer overall survival, earlier metastasis, and increased brain metastasis. In vitro, recombinant sEcad and stable sEcad overexpression in IBC cell lines promoted invasion, resistance to anoikis, and activation of pro-survival NF-κβ signaling. In vivo, mice injected with sEcad-overexpressing IBC cells had increased metastatic burden and reduced overall and brain metastasis-free survival. Further, sEcad induced reactive astrocytosis through the CXCL1/CXCL8-CXCR2 axis, and treatment with a brain-permeable CXCR2 antagonist reduced metastatic burden and prolonged survival in the brain metastasis models. CONCLUSION:sEcad drives brain metastasis by promoting invasion and anoikis resistance in cancer cells and inducing an inflammatory brain microenvironment via a targetable CXCL1/CXCL8-CXCR2 axis. These findings uncover a novel and critical role for sEcad and highlight CXCR2 as a therapeutic target in patients with metastatic IBC.

Background: Brain metastasis is a frequent site of relapse in patients with inflammatory breast cancer (IBC) - a rare, highly aggressive and metastatic variant of breast cancer. We have discovered that soluble E-cadherin (sEcad), an 80-kDa proteolytic fragment of full-length E-cadherin, correlated with increased risk of brain metastasis and death in patients with metastatic IBC. Additionally, we demonstrated that sEcad promotes brain metastasis growth and progression in HER2+ and triple-negative IBC brain metastasis models. However, how sEcad promotes brain metastatic progression is unknown. We hypothesize that sEcad promotes the production of specific cytokines, which, upon extracellular release, promote the activation of astrocytes, priming the brain microenvironment for metastatic growth. Methods: Stable overexpression of sEcad in IBC cell lines (MDA-IBC3 (ER–/HER2+) and SUM149 (ER–/HER2–) was achieved using lentiviral vectors. We injected MDA-IBC3-sEcad and control cells (tail-vein) and SUM149-sEcad and control cells (intracardiac) into SCID/Beige mice to assess brain metastasis burden and survival in mice. Human cytokine array was used to examine conditioned medium from sEcad high and control cells. Clinical datasets were used to compare expression and percent risk of brain relapse. Mice were treated with brain-permeable CXCR2 inhibitor in both IBC brain metastasis models. Multiplex quantitative imaging was used to visualize cells of the brain metastatic microenvironment. Results: Higher serum sEcad levels were significantly associated with reduced OS, earlier metastasis onset, and increased brain metastasis incidence. sEcad is an independent predictor of OS on multivariate analysis (hazard ratio [HR]=2.07 [95% CI 1.19-3.60], p=0.01). Treatment of astrocytes with recombinant sEcad increased reactive astrocytosis, in vitro and in vivo. Cytokine array analysis showed increased levels of pro-inflammatory cytokine CXCL1, DKK1 and CXCL8 in conditioned medium from sEcad-overexpressing IBC cells compared with control cells, which was validated by ELISA. In patient samples, CXCL1, CXCL8 and CXCR2, the receptor for CXCL1 and CXCL8, were expressed higher in brain metastasis compared to other metastases. Additionally, patients with high CXCL1/CXCL8 or CXCR2 expression had reduced brain metastasis relapse. Inhibition of CXCR2 decreased sEcad-mediated induction of reactive astrocytes. Treatment of mice bearing MDA-IBC3-sEcad and SUM149-sEcad brain metastases with the brain permeable CXCR2 inhibitor reduced number of brain metastasis, metastasis burden and prolonged survival of mice. Multiplexed immunofluorescence staining showed a significant reduction of reactive astrocytes in brain metastasis lesions treated with the CXCR2 inhibitor. Conclusion: Our findings underscore that sEcad drives brain metastasis by promoting an inflammatory brain microenvironment via a targetable CXCL1/CXCL8-CXCR2 axis. Targeting this axis presents a promising therapeutic strategy to effectively block brain metastasis in aggressive breast cancers. Citation Format: Xiaoding Hu, Yun Xiong, Emilly S Villodre, Juhee Song, Maria Stenkamp, Natalie Fowlkes, Elizabeth Leigh, Jeffery, Savitri Krishnamurthy, Junjie Chen, Wendy A Woodward, Debu Tripathy, Bisrat G Debeb. Targeting CXCL1-CXCR2 axis blocks brain metastasis in inflammatory breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS14-08.

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.

Brain metastasis is a frequent site of relapse in patients with inflammatory breast cancer (IBC) - a rare, highly aggressive variant of breast cancer. We discovered that soluble E-cadherin (sEcad), an 80-kDa E-cadherin fragment, correlates with increased brain metastasis risk and mortality in metastatic IBC patients. We further demonstrated that sEcad drives brain metastasis in HER2+ and triple-negative IBC mouse models. We hypothesize that sEcad induces specific cytokine production, activating astrocytes and priming the brain microenvironment for metastatic growth. sEcad-high and control IBC cells were injected into SCID/Beige mice to assess brain metastasis burden and survival. Human cytokine array analyzed conditioned medium from sEcad high and control cells. Clinical datasets compared expression and percent brain relapse rates. A brain-permeable CXCR2 inhibitor was tested for efficacy in IBC brain metastases. Multiplex quantitative imaging visualized cells in the brain metastatic microenvironment. Higher serum sEcad levels were independently correlated with reduced overall survival and increased brain metastasis. Cytokine array and ELISA showed increased levels of pro-inflammatory cytokines CXCL1, DKK1 and CXCL8 in conditioned medium from sEcad-overexpressing IBC cells compared with control cells. In patient samples, CXCL1, CXCL8 and CXCR2, the receptor for CXCL1 and CXCL8, were expressed higher in brain metastases and had increased brain metastasis relapse. sEcad promoted reactive astrocytosis in vitro and in vivo, while CXCR2 inhibition reduced this effect. Treating sEcad-high brain metastases with a brain- permeable CXCR2 inhibitor reduced metastasis, lowered burden and prolonged survival in two IBC mouse models. Multiplexed immunofluorescence showed fewer reactive astrocytes in brain metastasis lesions treated with the CXCR2 inhibitor. sEcad drives brain metastasis by promoting an inflammatory brain microenvironment via a targetable CXCL1/CXCL8-CXCR2 axis. Targeting this axis presents a promising therapeutic strategy to block brain metastasis in aggressive breast cancers.

The brain is a common site of relapse in inflammatory breast cancer (IBC), an E-cadherin positive, aggressive form of breast cancer. We found that elevated serum levels of soluble E-cadherin (sEcad), an 80-kDa fragment of E-cadherin, in patients with metastatic IBC correlated with poorer outcomes and increased rates of brain metastases. In our effort to understand the underlying mechanism, we discovered that sEcad binds to XIAP, an inhibitor of cell death, activating the pro-survival NF-kβ signaling in tumor cells. We also discovered that sEcad affects the tumor cell microenvironment by enhancing cancer cell adhesion to endothelial cells and inducing reactive astrocytosis in the brain. In addition, we found that sEcad-mediated reactive astrocytosis relies on the CXCL1/CXCL8-CXCR2 axis and treatment with a brain-permeable CXCR2 antagonist reduced brain metastatic burden and prolonged survival. These findings implicate sEcad in brain metastasis and provide new insights into potential therapeutic targets for IBC. Highlights:High serum sEcad levels correlate clinically with poor survival outcomes and development of brain metastasissEcad drives IBC brain metastasis growth in mouse modelssEcad binds XIAP to activate NFkB and promote anoikis resistance and invasion of IBC cells sEcad activates reactive astrocytes and induces CXCR2 expression on tumor cells in vitro and in vivo CXCR2-IN-1, a brain-permeable CXCR2 antagonist, reduces metastasis and improves survival in IBC brain metastasis models.

Background: Inflammatory breast cancer (IBC) is a rare, aggressive form of breast cancer but accounts for 10% of breast cancer-related deaths. Cancer stem cells (CSCs; tumor stemness) play a key role in tumor dormancy, progression, and treatment resistance, yet mechanisms that drive CSCs remain poorly defined. We recently identified that NDRG1 promotes tumor growth and progression in IBC mouse models, and that its depletion inhibits AKT phosphorylation. We hypothesized that NDRG1 is a key regulator of tumor stemness in IBC via activating AKT signaling. Methods: CSCs were assessed using surrogate markers including CD44+/CD24-, mammospheres, and in vivo limiting dilution assay experiments. To identify which AKT isoforms (AKT1, AKT2, or AKT3) or upstream kinases (SGK1, GSK3β) mediate NDRG1-induced CSCs, NDRG1-depleted cells were transfected with NDRG1 WT, AKT plasmids or SGK1/GSK3β phospho-site mutants or siRNA for SGK1/GSK3β. Results: NDRG1 depletion significantly reduced CD44+/CD24- subpopulation and mammosphere formation (p<0.001). Limiting dilution experiments demonstrated a significant reduction in tumor incidence and stemness frequency in mice transplanted with NDRG1 knockdown cells (p= 1 x 10-12). Each of the three AKT isoforms partially rescued tumor stemness in the NDRG1 knockdown IBC cells [CD44+/CD24-: NDRG1 KD vs AKT1 OE, p=0.008; vs AKT2 OE, p=0.005; vs AKT3 OE, p=0.001)]. Overexpression of known inactive SGK1 phospho-site mutants of NDRG1 restored tumor stemness in NDRG1-depleted cells (p≤0.005). While silencing GSK3β reduced CSCs in IBC cells SGK1 did not affect this subpopulation, indicating that the tumor stemness effect of NDRG1 is independent of SGK1 and its kinase activity and is regulated by GSK3β. Conclusions: Our findings underscore the critical role of NDRG1 as a regulator of IBC tumor stemness. We observed all three isoforms of AKT restored CSCs in NDRG1-depleted breast cancer cells, highlighting the significance of the NDRG1-AKT axis in governing stemness and tumor progression in IBC. Citation Format: Emilly Schlee Villodre, Xiaoding Hu, Wendy A. Woodward, Stefan Pusch, Debu Tripathy, Bisrat G. Debeb. NDRG1-AKT signaling promotes tumor stemness in inflammatory breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-06-23.

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

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

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. 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.

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.