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Tumor-associated macrophages (TAM) are among the most abundant immune cells in the tumor microenvironment and are important mediators of tumor development and progression. Here, we identified a macrophage-associated long noncoding RNA (lncRNA), MALR, that facilitates progression of esophageal squamous cell carcinoma (ESCC). TAM-mediated secretion of TNFα drove MALR upregulation in ESCC cells. MALR promoted aerobic glycolytic activity and facilitated angiogenesis by activating the HIF1α signaling pathway. Mechanistically, MALR bound the dsRBD1 domain of interleukin enhancer-binding factor 3 (ILF3), promoting ILF3 protein stability and ILF3-mediated liquid–liquid phase separation (LLPS), thereby enhancing HIF1α mRNA stability by preventing PARN-mediated degradation. Loss of MALR suppressed cell line–based and patient-derived xenograft tumor growth. Clinically, high expression of MALR positively correlated with HIF1α target gene expression and indicated poor prognoses for patients with esophageal cancer. Overall, this study uncovers the physiologic roles of MALR/ILF3-mediated LLPS in tumor microenvironment remodeling, highlighting the MALR–ILF3–HIF1α axis as a potential target for cancer therapy.Significance:Secretion of TNFα by tumor-associated macrophages stimulates cancer cells to upregulate lncRNA MALR, which induces ILF3 liquid–liquid phase separation and activation of HIF1α signaling to promote cancer progression.

Supplementary Table 2

Supplementary Methods

Supplementary Figures

Supplementary Table 1

Supplementary Figure from A Randomized, Open-Label, Multicenter, Phase 3 Study of High-Dose Vitamin C Plus FOLFOX ± Bevacizumab versus FOLFOX ± Bevacizumab in Unresectable Untreated Metastatic Colorectal Cancer (VITALITY Study)

Supplementary Data from A Randomized, Open-Label, Multicenter, Phase 3 Study of High-Dose Vitamin C Plus FOLFOX ± Bevacizumab versus FOLFOX ± Bevacizumab in Unresectable Untreated Metastatic Colorectal Cancer (VITALITY Study)

Purpose: To compare napabucasin (generator of reactive oxygen species) plus paclitaxel with paclitaxel only in patients with second-line advanced gastric or gastroesophageal junction (GEJ) adenocarcinoma. Patients and Methods: In the double-blind, phase III BRIGHTER study (NCT02178956), patients were randomized (1:1) to napabucasin (480 mg orally twice daily) plus paclitaxel (80 mg/m2 i.v. weekly for 3 of 4 weeks) or placebo plus paclitaxel. The primary endpoint was overall survival (OS). Secondary endpoints included progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), and safety. Results: Overall, 714 patients were randomized (napabucasin plus paclitaxel, n = 357; placebo plus paclitaxel, n = 357). 72.1% were male, 74.6% had gastric adenocarcinoma, and 46.2% had peritoneal metastases. The study was unblinded following an interim analysis at 380 deaths. The final efficacy analysis was performed on 565 deaths (median follow-up, 6.8 months). No significant differences were observed between napabucasin plus paclitaxel and placebo plus paclitaxel for OS (6.93 vs. 7.36 months), PFS (3.55 vs. 3.68 months), ORR (16% vs. 18%), or DCR (55% vs. 58%). Grade ≥3 adverse events occurred in 69.5% and 59.7% of patients administered napabucasin plus paclitaxel and placebo plus paclitaxel, respectively, with grade ≥3 diarrhea reported in 16.2% and 1.4%, respectively. Conclusions: Adding napabucasin to paclitaxel did not improve survival in patients with pretreated advanced gastric or GEJ adenocarcinoma. Consistent with previous reports, the safety profile of napabucasin was driven by manageable gastrointestinal events; grade ≥3 diarrhea occurred at a higher frequency with napabucasin plus paclitaxel versus placebo plus paclitaxel.

Suppression of gluconeogenesis elevates glycolysis and is commonly observed in tumors derived from gluconeogenic tissues including liver and kidney, yet the definitive regulatory mechanism remains elusive. Here, we screened an array of transcription regulators and identified the enhancer of zeste homolog 2 (EZH2) as a key factor that inhibits gluconeogenesis in cancer cells. Specifically, EZH2 repressed the expression of a rate-limiting gluconeogenic enzyme fructose-1, 6-bisphosphatase 1 (FBP1) and promoted tumor growth primarily through FBP1 suppression. Furthermore, EZH2 was upregulated by genotoxins that commonly induce hepatic and renal tumorigenesis. Genotoxin treatments augmented EZH2 acetylation, leading to reduced association between EZH2 and its E3 ubiquitin ligase SMURF2. Consequently, EZH2 became less ubiquitinated and more stabilized, promoting FBP1 attenuation and tumor formation. Intriguingly, FBP1 physically interacted with EZH2, competed for EZH2 binding, and dissembled the polycomb complex. Therefore, FBP1 suppresses polycomb-initiated transcriptional responses and constitutes a double-negative feedback loop indispensable for EZH2-promoted tumorigenesis. Finally, EZH2 and FBP1 levels were inversely correlated in tumor tissues and accurately predicted patient survival. This work reveals an unexpected cross-talk between epigenetic and metabolic events, and identifies a new feedback circuitry that highlights EZH2 inhibitors as liver and kidney cancer therapeutics.Significance:A novel feedback loop involving EZH2 and suppression of the gluconeogenesis enzyme FBP1 promotes hepatocellular cancer growth.See related commentary by Leithner, p. 657

Table S1. Primers sequence for qPCR Table S2. The correlation between clinicopathological parameters and ME1 expression Table S3. Univariate and multivariate analyses of various potential prognostic factors in GC patients

PDF file 18K, Bmal1 overexpression in the HCT116 and THC8307 cells after 30 generations of cell culture

Supplementary Figure from Randomized, Double-Blind, Placebo-Controlled Phase III Study of Paclitaxel ± Napabucasin in Pretreated Advanced Gastric or Gastroesophageal Junction Adenocarcinoma

Supplementary tables S1-S4 show analytic details of the ENCODE ChIP-Seq data and TCGA RNA-Seq data, together with primer sequences used in this study.

Supplemental Figure S1. The over-expression of CBX4, but not of other CBX members, suppresses the migration of DLD1 cells. Supplementary Figure S2. The knockdown of CBX4 enhances migration and invasion of CRC cells. Supplementary Figure S3. The over-expression of CBX4 inhibits the cell migration and invasion of CRC cells. Supplementary Figure S4. CBX4 represses the liver metastasis of HCT116 cells. Supplementary Figure S5. CBX4 is negatively related to the Runx2 expression in CRC cell lines. Supplementary Figure S6. The knockdown of Runx2 decreases the expression of Slug. Supplementary Figure S7. The promotion of cell migration and invasion after CBX4 knockdown primarily depends on Runx2. Supplementary Figure S8. The knockdown of Slug also reverses the promotion of cell migration and invasion after CBX4 knockdown. Supplementary Figure S9. IHC antibodies specifically recognized CBX4 or Runx2 protein. Supplementary Figure S10. The ΔSIM1/2 and CDM mutations marginally affect the functions of CBX4 in CRC. Supplementary Figure S11. CBX4 strongly binds with HDAC1, HDAC2, HDAC3 and HDAC9. Supplementary Figure S12. HDAC1, HDAC2, and HDAC3, but not HDAC9, are expressed in the tested CRC cell lines. Supplementary Figure S13. The ΔSIM1/2 and CDM mutations marginally affect the interaction of CBX4 with HDAC3. Supplementary Figure S14. The binding affinity of HDAC3 with the Runx2 promoter was dependent on CBX4. Supplementary Figure S15. The knockdown of HDAC3 abrogates the functions of CBX4 in CRC. Supplementary Figure S16. Determination of CBX4 fragments interacting with HDAC3. Supplementary Figure S17. The C-terminal 10 amino acids of CBX4 are responsible for its interaction with Bmi1. Supplementary Figure S18. The ΔDM1-CBX4 and ΔTM-CBX4 mutations, but not the deltaC-CBX4 mutation, abolish the inhibition of cell migration and invasion by CBX4.

The transcriptome analysis of HCT116 cells transfected with scrambled or CBX4 shRNA.

Table S1 is the correlation between CBX4 expression and Runx2 expression of CRC patients, Table S2 is correlation between CBX4 expression and clinicopathologic characteristics of CRC patients, and Table S3 is univariate and multivariate analysis for overall survival.

Purpose: Metastatic colorectal cancer patients with low epidermal growth factor receptor (EGFR) gene copy number are unlikely to respond to anti-EGFR monoclonal antibody (mAb) treatment. The objective of this study was to investigate EGFR fluorescence in situ hybridization (FISH) patterns of chromosome 7 disomy with efficacy of cetuximab therapy in metastatic colorectal cancer patients.Experimental Design: We detected the EGFR FISH patterns and KRAS status in 74 tumors from cetuximab-treated metastatic colorectal cancer patients and analyzed with response rate (RR) and progression-free survival (PFS).Results: One of the 16 (6.25%) patients with chromosome 7 homogeneous disomy (defined as FISH negative) had objective response to cetuximab. A total of 53(76.8%) patients with chromosome 7 pattern of variable ratios of disomy versus polysomy (defined as FISH positive) had a significantly higher RR (37.7% versus 6.25%; P = 0.01), a trend towards longer PFS (4.5 versus 2.9 months; P = 0.07). Among 54 KRAS wild-type patients, EGFR FISH-positive patients had significantly higher RR (51.3% versus 9%; P = 0.01) and longer PFS (5.0 versus 2.3 months; P = 0.02) than EGFR FISH-negative patients. However, among 20 KRAS mutant-type patients, there was no difference in RR (0% versus 0%) and PFS (2.5 versus 3.8 months; P = 0.51) between EGFR FISH-positive and -negative patients.Conclusion: Our results show firstly that patients with EGFR FISH pattern of chromosome 7 disomy have a very low chance to benefit from cetuximab-based therapy. EGFR FISH pattern of chromosome 7 disomy may be as a negative predicative factor for cetuximab response in KRAS wild-type metastatic colorectal cancer patients. Clin Cancer Res; 17(2); 382–90. ©2011 AACR.

Polycomb chromobox (CBX) proteins participate in the polycomb repressive complex (PRC1) that mediates epigenetic gene silencing and endows PRC1 with distinct oncogenic or tumor suppressor functions in a cell-type–dependent manner. In this study, we report that inhibition of cell migration, invasion, and metastasis in colorectal carcinoma requires CBX4-mediated repression of Runx2, a key transcription factor that promotes colorectal carcinoma metastasis. CBX4 inversely correlated with Runx2 expression in colorectal carcinoma tissues, and the combination of high CBX4 expression and low Runx2 expression significantly correlated with overall survival, more so than either CBX4 or Runx2 expression alone. Mechanistically, CBX4 maintained recruited histone deacetylase 3 (HDAC3) to the Runx2 promoter, which maintained a deacetylated histone H3K27 state to suppress Runx2 expression. This function of CBX4 was dependent on its interaction with HDAC3, but not on its SUMO E3 ligase, its chromodomain, or the PRC1 complex. Disrupting the CBX4–HDAC3 interaction abolished Runx2 inhibition as well as the inhibition of cell migration and invasion. Collectively, our data show that CBX4 may act as a tumor suppressor in colorectal carcinoma, and strategies that stabilize the interaction of CBX4 with HDAC3 may benefit the colorectal carcinoma patients with metastases. Cancer Res; 76(24); 7277–89. ©2016 AACR.

Genomic alterations of tumor suppressorsoften encompass collateral protein-coding genes that create therapeutic vulnerability to further inhibition of their paralogs. Here, we report that malic enzyme 2 (ME2) is frequently hemizygously codeleted with SMAD4 in gastric cancer. Its isoenzyme ME1 was upregulated to replenish the intracellular reducing equivalent NADPH and to maintain redox homeostasis. Knockdown of ME1 significantly depleted NADPH, induced high levels of reactive oxygen species (ROS), and ultimately cell apoptosis under oxidative stress conditions, such as glucose starvation and anoikis, in ME2-underexpressed cells. Moreover, ME1 promoted tumor growth, lung metastasis, and peritoneal dissemination of gastric cancer in vivo. Intratumoral injection of ME1 siRNA significantly suppressed tumor growth in cell lines and patient-derived xenograft–based models. Mechanistically, ME1 was transcriptionally upregulated by ROS in an ETV4-dependent manner. Overexpression of ME1 was associated with shorter overall and disease-free survival in gastric cancer. Altogether, our results shed light on crucial roles of ME1-mediated production of NADPH in gastric cancer growth and metastasis.Significance: These findings reveal the role of malic enzyme in growth and metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/8/1972/F1.large.jpg. Cancer Res; 78(8); 1972–85. ©2018 AACR.

Supplementary Table from Randomized, Double-Blind, Placebo-Controlled Phase III Study of Paclitaxel ± Napabucasin in Pretreated Advanced Gastric or Gastroesophageal Junction Adenocarcinoma

Figure legends for Figure S1-7.

PDF file 59K, Clinicopathological characteristics of patient samples and expression of Bmal1 in colorectal cancer

Supplementary Data from Randomized, Double-Blind, Placebo-Controlled Phase III Study of Paclitaxel ± Napabucasin in Pretreated Advanced Gastric or Gastroesophageal Junction Adenocarcinoma

Addtional information on shRNA sequences, qRT-PCR primers, promoter cloning primers, antibodies and ChIP-qPCR primers used in this study.

Supplementary Figure S1. SMAD4 and ME2 copy number are frequently altered in human malignancies. Supplementary Figure S2. ME1 maintains cell survival during glucose deprivation. Supplementary Figure S3. Effects of ME1 on epithelial mesenchymal transition in gastric cancer. Supplementary Figure S4. Simultaneous knockdown of ME1 and ME2 in HGC27 cells significantly suppressed tumor growth in vivo. Supplementary Figure S5. Immunoblots show increased ME1 and decreased ME2 expression in the tumor tissues compared with corresponding normal tissues of PDX models. Supplementary Figure S6. ETV4 regulates ME1 expression in gastric cancer. Supplementary Figure S7. Clinical significance of ETV4/ME1 axis in gastric cancer.

Purpose: The circadian clock gene Bmal1 is involved in cancer cell proliferation and DNA damage sensitivity. The aim of this study was to explore the effect of Bmal1 on oxaliplatin sensitivity and to determine its clinical significance in colorectal cancer.Experimental Design: Three colorectal cancer cell lines, HCT116, THC8307 and HT29, were used. The Bmal1-mediated control of colorectal cancer cell proliferation was tested in vitro and in vivo. MTT and colony formation assays were performed to determine the sensitivity of colorectal cancer cells to oxaliplatin. Flow cytometry was used to examine changes in the cell-cycle distribution and apoptosis rate. Proteins expressed downstream of Bmal1 upon its overexpression were determined by Western blotting. Immunohistochemistry was used to analyze Bmal1 expression in 82 archived colorectal cancer tumors from patients treated with oxaliplatin-based regimens.Results: Bmal1 overexpression inhibited colorectal cancer cell proliferation and increased colorectal cancer sensitivity to oxaliplatin in three colorectal cancer cell lines and HCT116 cells model in vivo. Furthermore, the overall survival of patients with colorectal cancer with high Bmal1 levels in their primary tumors was significantly longer than that of patients with low Bmal1 levels (27 vs. 19 months; P = 0.043). The progression-free survival of patients with high Bmal1 expression was also significantly longer than that of patients with low Bmal1 expression (11 vs. 5 months; P = 0.015). Mechanistically, the effect of Bmal1 was associated with its ability to regulate G2–M arrest by activating the ATM pathway.Conclusion: Bmal1 shows the potential as a novel prognostic biomarker and may represent a new therapeutic target in colorectal cancer. Clin Cancer Res; 20(4); 1042–52. ©2013 AACR.

PDF file 38K, The specific Bmal1 protein expression by Western blotting for each transformed cell line compared with its control cell line

Purpose:To compare napabucasin (generator of reactive oxygen species) plus paclitaxel with paclitaxel only in patients with second-line advanced gastric or gastroesophageal junction (GEJ) adenocarcinoma.Patients and Methods:In the double-blind, phase III BRIGHTER study (NCT02178956), patients were randomized (1:1) to napabucasin (480 mg orally twice daily) plus paclitaxel (80 mg/m2 i.v. weekly for 3 of 4 weeks) or placebo plus paclitaxel. The primary endpoint was overall survival (OS). Secondary endpoints included progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), and safety.Results:Overall, 714 patients were randomized (napabucasin plus paclitaxel, n = 357; placebo plus paclitaxel, n = 357). 72.1% were male, 74.6% had gastric adenocarcinoma, and 46.2% had peritoneal metastases. The study was unblinded following an interim analysis at 380 deaths. The final efficacy analysis was performed on 565 deaths (median follow-up, 6.8 months). No significant differences were observed between napabucasin plus paclitaxel and placebo plus paclitaxel for OS (6.93 vs. 7.36 months), PFS (3.55 vs. 3.68 months), ORR (16% vs. 18%), or DCR (55% vs. 58%). Grade ≥3 adverse events occurred in 69.5% and 59.7% of patients administered napabucasin plus paclitaxel and placebo plus paclitaxel, respectively, with grade ≥3 diarrhea reported in 16.2% and 1.4%, respectively.Conclusions:Adding napabucasin to paclitaxel did not improve survival in patients with pretreated advanced gastric or GEJ adenocarcinoma. Consistent with previous reports, the safety profile of napabucasin was driven by manageable gastrointestinal events; grade ≥3 diarrhea occurred at a higher frequency with napabucasin plus paclitaxel versus placebo plus paclitaxel.

PDF file 287K, Bmal1 overexpression increases apoptosis in HCT116 (A), THC8307 (B) and HT29 (C) cells after 48 h oxaliplatin treatment

Supplementary Table S1.

Supplementary Figures S1-S6 describe additional data to support the EZH2-FBP1 feedback circuitry in hepatic and renal cells upon genotoxin-induced tumorigenesis. Supplementary Materials and Methods provide experimental details on standard assays using commercial reagents and kits.

PDF file 17K, Bmal1 protein expression in different CRC cell lines

Long noncoding RNAs (lncRNA) are involved in tumorigenesis and drug resistance. However, the roles and underlying mechanisms of lncRNAs in colorectal cancer are still unknown. In this work, through transcriptomic profiling analysis of 21 paired tumor and normal samples, we identified a novel colorectal cancer–related lncRNA, MNX1-AS1. MNX1-AS1 expression was significantly upregulated in colorectal cancer and associated with poor prognosis. In vitro and in vivo gain- and loss-of-function experiments showed that MNX1-AS1 promotes the proliferation of colorectal cancer cells. MNX1-AS1 bound to and activated Y-box-binding protein 1 (YB1), a multifunctional RNA/DNA-binding protein, and prevented its ubiquitination and degradation. A marked overlap between genes that are differentially expressed in MNX1-AS1 knockdown cells and transcriptional targets of YB1 was observed. YB1 knockdown mimicked the loss of viability phenotype observed upon depletion of MNX1-AS1. In addition, MYC bound the promoter of the MNX1-AS1 locus and activated its transcription. In vivo experiments showed that ASO inhibited MNX1-AS1, which suppressed the proliferation of colorectal cancer cells in both cell-based and patient-derived xenograft models. Collectively, these findings suggest that the MYC–MNX1-AS1–YB1 axis might serve as a potential biomarker and therapeutic target in colorectal cancer. Significance: This study highlights the discovery of a novel colorectal cancer biomarker and therapeutic target, MNX1-AS1, a long noncoding RNA that drives proliferation via a MYC/MNX1-AS1/YB1 signaling pathway.

Suppression of gluconeogenesis elevates glycolysis and is commonly observed in tumors derived from gluconeogenic tissues including liver and kidney, yet the definitive regulatory mechanism remains elusive. Here, we screened an array of transcription regulators and identified the enhancer of zeste homolog 2 (EZH2) as a key factor that inhibits gluconeogenesis in cancer cells. Specifically, EZH2 repressed the expression of a rate-limiting gluconeogenic enzyme fructose-1, 6-bisphosphatase 1 (FBP1) and promoted tumor growth primarily through FBP1 suppression. Furthermore, EZH2 was upregulated by genotoxins that commonly induce hepatic and renal tumorigenesis. Genotoxin treatments augmented EZH2 acetylation, leading to reduced association between EZH2 and its E3 ubiquitin ligase SMURF2. Consequently, EZH2 became less ubiquitinated and more stabilized, promoting FBP1 attenuation and tumor formation. Intriguingly, FBP1 physically interacted with EZH2, competed for EZH2 binding, and dissembled the polycomb complex. Therefore, FBP1 suppresses polycomb-initiated transcriptional responses and constitutes a double-negative feedback loop indispensable for EZH2-promoted tumorigenesis. Finally, EZH2 and FBP1 levels were inversely correlated in tumor tissues and accurately predicted patient survival. This work reveals an unexpected cross-talk between epigenetic and metabolic events, and identifies a new feedback circuitry that highlights EZH2 inhibitors as liver and kidney cancer therapeutics. Significance: A novel feedback loop involving EZH2 and suppression of the gluconeogenesis enzyme FBP1 promotes hepatocellular cancer growth. See related commentary by Leithner, p. 657

Background: Biomarkers that could predict the beneficial effects of immune checkpoint inhibitors (ICIs) in combination with chemotherapy in treatment-naive advanced or recurrent gastric or gastroesophageal junction (G/GEJ) adenocarcinoma patients are lacking. Methods: We conducted a retrospective analysis of patients with G/GEJ adenocarcinoma who had undergone first-line treatment with programmed death-1 (PD-1) inhibitor plus chemotherapy between October 2017 and July 2021. Comparative analysis of the objective response rate (ORR) was performed using the Chi-square or Fisher’s exact tests. Survival curves were plotted by the Kaplan-Meier analysis and compared for significance with a log-rank test. Univariate and multivariate Cox proportional hazard models were established for evaluating the prognostic value of clinicopathological factors. Results: Of the 172 enrolled patients, 142 showed measurable lesions. The ORR was 52.8%. Higher response rates were observed in patients with age greater than or equal to 60 (p = 0.013), non-diffuse type (p < 0.001), synchronous metastasis (p = 0.029), distant lymph node metastasis (p = 0.002), non-peritoneal metastasis (p= 0.002), HER2 positivity (p = 0.002), and PD-L1 CPS greater than or equal to 5 (p = 0.017). Through univariate analysis, age, histology, number of metastatic sites, and peritoneal metastasis were found to be associated with progression-free survival (PFS). Through multivariate analysis, peritoneal metastasis occurrence was the only identified independent indicator of poor PFS (HR 2.768, 95% CI 1.184-6.472, p = 0.019). Additionally, multi-factor combination results showed that patients with at least one of the following: mismatch repair-deficient (D-MMR)/microsatellite instability-high (MSI-H), HER2 (+), EBV (+), and/or PD-L1 CPS greater than or equal to 5, exhibited higher response rates and longer PFS as compared to those lacking these factors; this benefit was consistent after excluding PD-L1 expression. Among the clinical combinatorial factors, significantly poor ORRs and lower PFS were observed in patients with age < 60, diffuse type, and peritoneal metastasis as compared to those without any of these factors. Conclusions: Peritoneal metastasis was an independent biomarker of poor efficacy to immunotherapy combined with chemotherapy in G/GEJ cancer patients in first-line setting. Some combination factors could be beneficial for efficacy prediction. Citation Format: Yu-Ting Sun, Shi-Xun Lu, Ming-Yu Lai, Xia Yang, Wen-Long Guan, Li-Qiong Yang, Yu-Hong Li, Feng-Hua Wang, Rui-Hua Xu, Miao-Zhen Qiu. Predictive biomarkers for the efficacy of PD-1 inhibitors plus chemotherapy for gastric/gastroesophageal junction cancer in first-line setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5092.

Background: Overall survival (OS) for advanced or metastatic HER2-negative GC/GEJC with standard 1L chemo remains poor (median OS < 1 year). CheckMate 649 is the largest randomized, global phase 3 study of 1L programmed death (PD)-1 inhibitor-based therapies in GC/GEJC/EAC (NCT02872116). Results from this study demonstrated superior OS, along with progression-free survival (PFS) benefit and an acceptable safety profile with 1L NIVO + chemo vs chemo alone (Moehler et al. Ann Oncol 2020). We present the pre-planned subgroup analysis from CheckMate 649 in Chinese pts.Methods: Adults with previously untreated, unresectable advanced or metastatic GC/GEJC/EAC were enrolled regardless of PD ligand 1 (PD-L1) expression. Pts with known HER2-positive status were excluded. Pts were randomized to receive NIVO (360 mg Q3W or 240 mg Q2W) + chemo (XELOX Q3W or FOLFOX Q2W), NIVO + ipilimumab, or chemo. Dual primary endpoints for NIVO + chemo vs chemo were OS and PFS by blinded central review in pts with PD-L1 combined positive score (CPS) ≥ 5.Results: 208 Chinese pts were concurrently randomized to NIVO + chemo or chemo, including 156 pts (75%) with PD-L1 CPS ≥ 5. Median age was 60 years; 88% had GC; 12% had GEJC; no pts had EAC. At minimum follow-up of 12 months (mo), NIVO + chemo demonstrated clinically meaningful improvement in OS and PFS vs chemo in pts with PD-L1 CPS ≥ 5 (OS, HR 0.54 [95% CI 0.36-0.79]; PFS, HR 0.52 [0.34-0.77]; Table), consistent with the overall study population. OS benefit was also observed in pts with PD-L1 CPS ≥ 1 and the all-randomized population (Table). No new safety signals were identified. Conclusions: NIVO + chemo demonstrated a clinically meaningful improvement in OS and PFS and an acceptable safety profile vs chemo alone in previously untreated Chinese pts, consistent with the overall study population with advanced GC/GEJC/EAC from CheckMate 649. TableEfficacyNIVO + chemoChemoPD-L1 CPS ≥ 5N = 75N = 81Median OS (95% CI), mo15.5 (11.9-25.5)9.6 (8.0-12.1)HR (95% CI)0.54 (0.36-0.79)Median PFS (95% CI), mo8.5 (5.9-12.4)4.3 (4.1-6.5)HR (95% CI)0.52 (0.34-0.77)ORR (95% CI), %63 (51-74)43 (32-55)PD-L1 CPS ≥ 1N = 89N = 94Median OS (95% CI), mo14.3 (11.5-17.5)9.9 (8.1-12.1)HR (95% CI)0.62 (0.43-0.87)All randomizedN = 99N = 109Median OS (95% CI), mo14.3 (11.5-17.5)10.3 (8.1-12.1)HR (95% CI)0.61 (0.44-0.85)Safety: Treatment-related adverse events, n (%)All treatedN = 99N = 106Any grade98 (99)100 (94)Grade 3-464 (65)53 (50)Leading to discontinuation50 (51)27 (25)Deaths1 (1)1 ( Citation Format: Lin Shen, Yuxian Bai, Xiaoyan Lin, Wei Li, Jufeng Wang, Xiaochun Zhang, Hongming Pan, Chunmei Bai, Li Bai, Ying Cheng, Jingdong Zhang, Haijun Zhong, Yi Ba, Wenwei Hu, Ruihua Xu, Weijian Guo, Shukui Qin, Nong Yang, Jianwei Lu, Kohei Shitara, Ming Lei, Mingshun Li, Nicole Bao, Tian Chen, Tianshu Liu. First-Line (1L) nivolumab (NIVO) plus chemotherapy (chemo) versus chemo in patients (pts) with advanced gastric cancer/gastroesophageal junction cancer/esophageal adenocarcinoma (GC/GEJC/EAC): CheckMate 649 Chinese subgroup analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT184.

Backgrounds: Tumor regression grade (TRG) reflects the chemoradiosensitivity of rectal cancer patients undergone neoadjuvant treatment, and relates to distinct probabilities of cancer recurrence and survival. However, in clinical practice, it is significantly challenging to rely solely on radiographic or clinical diagnostic information to obtain a patient's pathological response pre-treatment for treatment optimization. The aim was combining both the tumor information of macroscopic radiological and microscopic pathological images to develop and validated a more accurate signature for pretreatment prediction of TRG to neoadjuvant chemoradiotherapy (nCRT) in patients with locally advanced rectal cancer (LARC) on a multicenter dataset. Materials and Methods: We prospectively enrolled 981 patients (303 in the primary cohort, 678 in three external validation cohort) with clinicopathologically confirmed LARC treated with nCRT followed by surgery between Aug 2007 and Nov 2017, and collected their pretreatment multi-parametric MRI (mp-MRI), whole-slide image (WSI) of biopsy specimen, and pathologic response according to AJCC TRG system as well as clinical outcomes. Briefly, an artificial intelligence model integrating quantitative imaging features of mp-MRI and WSI was proposed to predict each nCRT treated patient into a particular 4-category TRG. The signature from the model (hereafter Rp-Grade) was further assessed in three independent validation cohort. Results: Rp-Grade yielded an overall ACC of 87.76% and revealed significant improvement than signature generating from model with mp-MRI or WSI features alone in all validation cohorts (P Performance of radiopathomics signature for predicting 4-category AJCC/CAP TRGMetrics (%) [95% CI]Validation Cohort1 (N=480)Validation Cohort2 (N=150)Validation Cohort3 (N=48)Accuracy87.76 [84.86-90.66]79.71 [72.52-84.9]81.24 [70.15-92.34]PPV (TRG=0)94.58 [90.48-98.69]93.52 [84.68-100.0]83.09 [61.19-100.0]PPV (TRG=1)92.02 [86.19-97.85]70.26 [56.42-84.1]70.26 [56.42-84.1]PPV (TRG=2)83.9 [79.6-88.19]74.24 [63.57-84.92]71.49 [52.04-90.94]PPV (TRG=3)-92.54 [78.21-100.0]-Sensitivity (TRG=0)96.49 [93.03-99.95]96.62 [89.92-100.0]91.3 [74.73-100.0]Sensitivity (TRG=1)96.49 [93.03-99.95]75.57 [61.75-89.4]63.39 [42.96-83.82]Sensitivity (TRG=2)97.16 [95.02-99.29]100.0 [100.0-100.0]100.0 [100.0 100.0]Sensitivity (TRG=3)-35.55 [19.99-51.12]-Note: The evaluation results were represented by ‘-' when the number of categories was insufficient in the independent cohort test. Conclusions: Combining the macroscopic radiological information of the whole tumor and microscopic pathological information of local lesions from biopsy, radiopathomics was a potential strategy for the pretreatment prediction of the TRG in patients with LARC who underwent nCRT. Citation Format: Lizhi Shao, Zhenyu Liu, Lili Feng, Xiaoying Lou, Zhenhui Li, Xiao-Yan Zhang, Xuezhi Zhou, Kai Sun, Da-Fu Zhang, Lin Wu, Guanyu Yang, Ying-Shi Sun, Ruihua Xu, Xiangbo Wan, Xinjuan Fan, Jie Tian. Radiopathomics strategy combining multiparametric MRI with whole-slide image for pretreatment prediction of tumor regression grade to neoadjuvant chemoradiotherapy in rectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2014.

Background/Aims: Small cell carcinoma of the esophagus (SCCE) is a rare and highly deadly malignancy with rapid disease progress and extensive metastasis, whereas present therapy for the cancer is limited, making the identification of a new treatment strategy an area of intense research. We previous reported the genomic characteristics and alterations of the SCCE, but the transcriptome abnormality of the cancer has not been investigated yet. Method: we performed an RNA Sequencing on nine paired samples from patients with SCCE and conducted a series of comprehensive analysis on the data. DESeq2 and GSEA were applied on transcriptome of SCCE tumor tissue and their para-normal to identify differential expressive genes and associated pathways. Besides, with MCP-counter and a list of immunity related genes, we also compared immune signal of the expression data in pair. By applying a rigorous batch removing strategy, including preprocessing raw data in identical method and RUVSeq, transcriptome data of healthy esophageal mucosa from GTEx was involved as control samples in comparison. In addition, we applied CIBERSORT to calculate the fraction of different tumor infiltrated leukocytes (TILs) of SCCE and compare the number with those of other cancers that have been proven to be potentially responsive to immunotherapy. Results: We identified 1486 and 1782 genes were significantly up-regulated and down-regulated in SCCE tumor tissue against their para-normal, respectively. Pathway analysis revealed the E2F Target and G2M checkpoint are significantly enriched in tumor tissue. Immune signal analysis across the samples in pair showed that in 3 out of 9 paired sample, immunity reaction upregulates in tumor tissue. T-SNE cluster across transcriptome data of SCCE tumor tissue, their para-normal and healthy esophageal mucosa showed the para-normal tissue, namely the normal tissue adjacent to tumor, is more similar to tumor tissue than the healthy. CIBERSORT analysis showed the difference in TILs fraction between SCCE and the compared cancers. For example, the relative fraction of plasma cell of SCCE is higher than those of ESCC(P=0.040), CIN type of STAD(P=0.040), while lower than those of SCLC(P=0.020). M2 macrophages is higher in SCCE than ESCC(P=0.002), EAC(P Conclusion: In summary, our analysis provides transcriptome evidence that immunotherapy might be an efficient treatment strategy for small cell esophagus cancer. Citation Format: Qi Zhao, Yan-xing Chen, Ying-nan Wang, Qi-nian Wu, Hui Sheng, JIa-jia Hu, Yun-xin Lu, Ze-xian Liu, Zhi-xiang Zuo, Feng Wang, Rui-hua Xu. Systematic transcriptome analysis of small cell carcinoma of esophagus suggests a possibility for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4242.

Background/Aims: Colorectal cancer (CRC) is a common cancer worldwide with increasing morbidity in China. Approximately 5% CRC are caused by well-defined hereditary CRC syndromes, whose inherited susceptibility account for nearly one-third of CRC predisposition. Most of hereditary CRC syndromes were diagnosed at the age of lower than 70, whereas little is known about the mutational profiles Chinese patients and their correlations with clinical features. Method: In our present research, we retrospectively collected 731 patients who were diagnosed as CRC from Sun Yat-sen University Cancer Center, with ages lower than 70. All of them received genetic mutation detection consisting of a set of 14 specific inherited genes, which used target area capture and the second-generation high throughput sequencing technology. The characteristics of he patients with or without mutations were compared. We applied several comparison analysis to investigate the germline mutations distribution of genes that associated with the defined hereditary CRC syndromes in Chinese cohort. Results: 397 patients (54.3%) harbored functionally variants of the 14 genes in CRC patients that diagnosis lower than 70. Intriguingly, we observed no statistical difference for clinical factors such as age, sex, smoke, drink, family history, pathology grade, clinic stage and overall survival between groups. In Mut group, the mutation frequency of each genes are MLH1(21.4%), MSH2(17.6%), MSH6(15.1%), PMS2(9.3%), EPCAM(8.3%), MLH3(8.1%), AXIN2(7.1%), PMS1(4.0%), MUTYH(12.8%), APC(12.3%), STK11(3.5%), BMPR1A(1.8%), SMAD4(0.3%), PTEN(1.0%). Genome mutual exclusivity analysis has identified a set of genes such as MLH1, MSH2, MUTYH, MSH6, APC and PMS2, for which most pairwise combinations are found rarely cooccurred. In correlation analysis between mutational genes and clinical features, pathology grade correlates strongly with PMS1 and SMAD4(P Conclusion: The clinic features of two groups have no statistic difference, which suggested other genes undiscovered may play a partial role in pathogenesis of patients of non-MUT group. In summary, we conducted a retrospective study to lead a comprehensive comparison of mutational profiles between MUT and non-MUT hereditary CRC patients, which supplemented the limited data of hereditary CRC in China. Citation Format: Feng Wang, Qi Zhao, Teng-jia Jiang, Ying-nan Wang, Fu-rong Liu, Jia-jia Hu, Pei-rong Ding, Zhi-zhong Pan, Jian-yong Shao, Rui-hua Xu. Germline mutational profileof Chinese patients with colorectal cancer and diagnosed lower than 70 years [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4161.

Purpose: Golgi phosphoprotein 3 (GOLPH3) has been reported to be involved in various biologic processes. The clinical significance and biologic role of GOLPH3 in breast cancer, however, remains unknown. Experimental Design: Expression of GOLPH3 in normal breast cells, breast cancer cells, and 6-paired breast cancer and adjacent noncancerous tissues were quantified using real-time PCR and Western blotting. GOLPH3 protein expression was analyzed in 258 archived, paraffin-embedded breast cancer samples using immunohistochemistry. The role of GOLPH3 in breast cancer cell proliferation and tumorigenicity was explored in vitro and in vivo. Western blotting and luciferase reporter analyses were used to investigate the effect of GOLPH3 overexpression and silencing on the expression of cell-cycle regulators and FOXO1 transcriptional activity. Results: GOLPH3 was significantly upregulated in breast cancer cells and tissues compared with normal cells and tissues. Immunohistochemical analysis revealed high expression of GOLPH3 in 133 of 258 (51.6%) breast cancer specimens. Statistical analysis showed a significant correlation of GOLPH3 expression with advanced clinical stage and poorer survival. Overexpression and ablation of GOLPH3 promoted and inhibited, respectively, the proliferation and tumorigenicity of breast cancer cells in vitro and in vivo. GOLPH3 overexpression enhanced AKT activity and decreased FOXO1 transcriptional activity, downregulated cyclin-dependent kinase (CDK) inhibitor p21Cip1, p27Kip1, and p57Kip2, and upregulated the CDK regulator cyclin D1. Conclusion: Our results suggest that high GOLPH3 expression is associated with poor overall survival in patients with breast cancer and that GOLPH3 overexpression increases the proliferation and tumorigenicity of human breast cancer cells. Clin Cancer Res; 18(15); 4059–69. ©2012 AACR.

Background & Aims—Small cell carcinoma of the oesophagus (SCCE) is a deadly malignancy but its genetic characteristics remain unknown and treatment is commonly adopted from therapies for its histologically identical counterpart, small cell lung cancer (SCLC). We conducted a large-scale genomic analysis of SCCE to determine the mutational landscape of this deadly disease. Methods—We performed whole-exome sequence of tumour and para normal esophageal tissues from 55 patients with SCCE who underwent surgery at seven institutions in China. Comprehensive analyses were carried out to identify key genetic events in SCCE, including significant mutations, copy number variation, pathway disruption. We also performed ultra-deep targeted sequencing on 20 specimens and microarray assays on 24 specimens to validate somatic mutations and copy number variations respectively. Using an unsupervised hierarchical clustering approach, the genomic characteristics of SCCE were compared with other types of malignancies including oesophageal squamous cell carcinoma, oesophageal adenocarcinoma and et al. In vitro proliferation and migration assays were conducted to assess the function of PDE3A. Results—The mutational spectrum of SCCE was dominated by C>T/G>A transitions and the mutational signatures classified the patients into 3 groups. We identified three novel significantly mutated genes (PDE3A, PTPRM and CBLN2) and mutations in five other well-known tumour-associated genes (TP53, RB1, NOTCH1, FAT1 and FBXW7). Non-silent alterations of chromatin remodeling genes were detected in 96.4%. Moreover, pathway enrichment analysis revealed that genes involved in the cell cycle, p53, Notch and Wnt signaling were frequently altered in SCCE. Mutations in NOTCH family correlated with shorter overall survival. Furthermore, comparison analysis of mutation signatures revealed that SCCE tumours were more closely related to oesophageal squamous cell carcinoma, rather than oesophageal adenocarcinoma and SCLC, suggesting that current therapies for SCCE may be inadequate. Functional characterizations suggested that PDE3A acts as a novel oncogene. Conclusions—Our findings reveal key somatic alterations in SCCE, and provide insights to better understand the pathogenesis of SCCE and to develop better treatment strategies for patients with the condition. Citation Format: Feng Wang, Dongbing Liu, Qi Zhao, Gang Chen, Xinming Liu, Yingnan Wang, Hong Su, Yanru Qin, Yifu He, Qingfeng Zou, Yanhui Liu, Youen Lin, Hui Sheng, Jiajia Hu, Zexian Liu, Zhixiang Zuo, Jinxin Bei, Ruihua Xu. The genomic landscape of small cell carcinoma of the oesophagus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2345.

Background Prognostic and predictive markers are needed to predict the clinical outcomes of patients with advanced colorectal cancer (CRC) who receive standard first-line treatments. We performed a prospective cohort study in patients with advanced CRC to identify a miRNA signature that could predict the benefit of receiving first-line chemotherapy for these patients. Methods Twenty-one paired tumours and adjacent normal tissues were collected from patients with advanced CRC and analysed by miRNA microarrays. Between tumour and normal tissues, 33 miRNAs were differentially expressed. The differential expression of these miRNAs was confirmed by qRT-PCR using paraffin-embedded specimens from another group of 76 patients from a prospective cohort study. A two-miRNA-based signature was obtained using the least absolute shrinkage and selection operator (LASSO) Cox regression model based on the association between the expression of each miRNA and the progression-free survival (PFS) time of individual patients. Internal and external validation cohorts, including 45 and 50 patients with advanced CRC, respectively, were performed to validate the prognostic and predictive accuracy of this signature. Results A signature based on two miRNAs, miR-125b-2-3p and miR-933, was built by the LASSO model. CRC patients were classified into high- and low-risk groups for disease progression based on this tool. The patients with high risk scores generally had worse PFS than those with lower risk scores. In the training set, the average PFS was 23.906 months for the low-risk group and 9.010 months for the high-risk group (hazard ratio [HR] 2.535, 95% CI 1.478-4.348, p=0.001). In the internal validation set, the average PFS was 14.029 months for the low-risk group and 6.334 months for the high-risk group (HR 3.173, 95% CI 1.415-7.116, p=0.005). In the external validation set, the average PFS was 16.815 months for the low-risk group and 7.657 months for the high-risk group (HR 2.498, 95% CI 1.281-4.871, p=0.007). Furthermore, we detected miR-125b-2-3p associated with CRC cell sensitivity to first-line chemotherapy. The two-miRNA-based signature was an independent prognostic factor for predicting the outcome of patients with metastatic CRC receiving first-line chemotherapy. Citation Format: Zhaolei Zeng, Jiahua Lu, Zhixiang Zuo, Qi Zhao, Ruihua Xu. MicroRNA expression analysis of advanced colorectal cancer reveals a microRNA signature with prognostic and predictive value [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5407.

Background: CT (fluorouracil/platinum) + H is standard of care (SoC) for 1L HER2-positive mGC/GEJC based on the ToGA Phase 3 study. ToGA post hoc analyses showed that lowest H trough concentration (Cycle 1 Ctrough) quartile pts had shorter overall survival (OS) than pts in higher quartiles, and that they were enriched for poorer prognostic factors at baseline, e.g. ECOG 2, no prior gastrectomy, ?2 metastatic sites. HELOISE (NCT01450696) investigated whether higher dose H (HD H) + CT, when compared to SoC H + CT, improves OS in 1L HER2-positive mGC/GEJC. Methods: Pts with the high risk features above were randomized 1:1 to loading dose H 8 mg/kg then SoC H maintenance 6 mg/kg every 3 weeks (q3w) or loading dose H 8 mg/kg then HD H maintenance 10 mg/kg q3w until progression; each arm combined with cisplatin 80 mg/m2 + capecitabine 800 mg/m2 in cycles 1-6. The primary objective was HD H OS superiority (target HR 0.75, all randomized pts); other objectives included pharmacokinetics (PK) and safety. Final results are from an interim analysis for futility at 125 OS events. Results: 248 pts had been randomized at clinical cutoff, Feb 13, 2015. Baseline characteristics/demographics were balanced overall, with median ages 58.5 and 62.6 years in the SoC H and HD H arms, respectively; 23% of pts per arm were female. Safety was comparable between arms, with no difference in congestive heart failure (0% each). The OS futility boundary was crossed (HR 1.24 > 0.95); safety, efficacy, and PK data are shown in the table. A marked increase in mean Ctrough was observed following the first HD H cycle vs SoC H. SoC H (8 mg/kg + 6 mg/kg) + CTHD H (8 mg/kg + 10 mg/kg) + CTPrimary tumorn=124n=124Gastric/GEJ, n (%)95 (76.6)/29 (23.4)101 (81.5)/23 (18.5)Treatmentn=124n=123Median H cycles/median capecitabine cycles/median cisplatin cycles, n6.5/6.0/6.06.0/6.0/5.0Safety, pts with ≥1:n=124n=123Any grade adverse event (AE), n (%)113 (91.1)112 (91.1)Serious AE (SAE)/SAE cardiac disorder, n (%)30 (24.2)/3 (2.4)35 (28.5)/4 (3.3)Efficacyn=124n=124OS events, n (%)58 (46.8%)67 (54.0%)Median OS, months12.510.6Stratified OS HR (95% CI)1.24 (0.86-1.78) Log-rank P=0.24Median PFS, months5.755.59Stratified PFS HR (95% CI)1.04 (0.76-1.40) Log-rank P=0.82PK: Mean Ctrough, μg/mL (% coefficient of variation)Cycle 1, 8 mg/kg loading H (n=101 and 99)17.1 (82.9)18.1 (100.1)Cycle 2, first dosing HD H (n=94 and 77)19.3 (45.7)35.6 (54.6)Cycle 7, steady-state H (n=51 and 44)31.4 (45.1)58.1 (47.5) Conclusions: Although HD H maintenance dosing is associated with higher H concentrations in HELOISE, we observed no increased efficacy (OS, PFS) and no new safety signals. HELOISE confirms SoC H (8 mg/kg loading dose followed by H 6 mg/kg q3w maintenance doses) as SoC with CT for 1L treatment of HER2-positive mGC/GEJC. Citation Format: Manish A. Shah, Ruihua Xu, Yung-Jue Bang, Paulo M. Hoff, Tianshu Liu, Luis A. Herraez-Baranda, Fan Xia, Amit Garg, Mona Shing, Josep Tabernero. HELOISE: phase IIIB randomized multicenter study comparing two trastuzumab (H) dose regimens combined with chemotherapy (CT) as first-line (1L) therapy in patients (pts) with HER2-positive metastatic gastric/gastroesophageal junction adenocarcinoma (mGC/GEJC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT108.

The tissue microenvironment promotes cancer cell survival and drug resistance, and thus presents a major challenge in cancer treatment. Recent study suggests that the microenvironmental protection of chronic lymphocytic leukemia (CLL) cells is particular important due to the unique metabolic properties of CLL cells. Emerging evidence suggests that altered fatty acid metabolism may be associated with the pathogenesis of CLL and could potentially provide a biochemical basis for targeting CLL. In the current study, we tested three classes of compounds that respectively inhibit the de novo fatty acids synthesis (FASN), block the transport of fatty acids from cytosol into mitochondria, or suppress fatty acid oxidation, for their ability to kill CLL cells in the presence and absence of bone marrow stromal cells. While all three inhibitors exhibited cytotoxic effect again CLL cells when the leukemia cells were culture alone, only the compound that inhibited the transport of fatty acids into mitochondria was highly effective in killing CLL in the presence of bone marrow stromal cells. Mechanistic study revealed that the cytotoxicity was not due to inhibition of ATP generation through mitochondrial β-oxidation of fatty acids, since there was no significant decrease in mitochondrial respiratory activity. We further showed that inhibition of FA transport into mitochondria suppressed biosynthesis of mitochondrial phospholipid, leading to a significant depletion of cardiolipin (CL), which is essential for maintaining mitochondrial membrane integrity. Depletion of CL by the FA transport inhibitor in CLL cells resulted in mitochondrial depolarization, release of cytochrome c, and massive apoptosis. Since this cell death process was through the intrinsic pathway and could not be rescued by the stromal environment, this might explain why inhibition of FA transport into the mitochondrial was highly effective in killing CLL cells in the presence of stromal cells. Importantly, we found that inhibition of FA transport had low toxicity in normal lymphocytes and stromal cells, suggesting that the normal cells might be less dependent on FA transport for cardiolipin biosynthesis. In summary, our study suggests that inhibition of FA transport into the mitochondria is a novel therapeutic strategy to effectively and selectively kill CLL cells in stromal microenvironment, and may potentially improve the clinical outcome of CLL treatment. Citation Format: Panpan Liu, Jinyun Liu, Marcia A. Ogasawara, Helene Pelicano, Ruihua Xu, Michael J. Keating, Peng Huang. A novel therapeutic strategy to effectively kill CLL cells in stromal microenvironment by targeting lipid metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2697. doi:10.1158/1538-7445.AM2014-2697

Glioblastoma multiforme (GBM) is a common and highly aggressive primary brain tumor with poor prognosis due in part to drug resistance and high incidence of tumor recurrence. The drug resistant and cancer recurrence phenotype may be ascribed to the presence of cancer stem cells, which seem to reside in special stem-cell niches in vivo and require special culture conditions including certain growth factors and serum-free medium to maintain their stemness in vitro. In this study, we used two lines of stem-like cancer cells (GSC11 and GSC23) derived from the tumor tissues of two GBM patients by classical neurosphere culture system, and showed that addition of serum (FBS) to the medium induced an increase of ROS, leading to aberrant differentiation and eventual cell death. Although both GBM stem-like cell lines showed high percentage of CD133-positive cells (20% in GSC11 and 70% in GSC23), exposure to serum only caused a moderate decrease of CD133 expression at later time points. Several methods were used to further test the role of ROS in affecting the stemness of the GSC cells and their self-renewal capacity. Addition of 5% FBS or removal of the antioxidant supplements (B27) from the culture medium caused the spheroid GSC cells to attach to the culture surface, exhibit differentiation morphology, decrease expression of certain stem-cell markers, and eventually die within two weeks. Serum exposure also resulted in a decrease of the GSC ability to form colonies in soft agar assay and to grow tumors in a mouse xenograft model. Although an increase of general cellular ROS could be detected when GSC cells were exposed to FBS, the mitochondrial superoxide appeared to be prominent and likely to be a primary event, which led to a compensatory increase of the mitochondrial SOD2 expression and upregulation of glutathione synthesis. Interestingly, addition of the antioxidant N-Acetylcysteine to the FBS-containing medium partially blocked the FBS-induced aberrant differentiation and improved cell viability and self-renewal capacity, suggesting that ROS may play an important role in affecting stemness and differentiation status of the cancer stem cells. We postulate that during tumor development, only the progeny cells that acquire the ability to counteract the impact of ROS increase induced by serum may eventually proliferate and grow as the tumor bulk, and that proper redox-modulation may be a potential novel strategy to block tumor formation and possibly kill the stem-like cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4266.