Search

Adoptive cell transfer (ACT) immunotherapy has remarkable efficacy against some hematologic malignancies. However, its efficacy in solid tumors is limited by the adverse tumor microenvironment (TME) conditions, most notably that acidity inhibits T and natural killer (NK) cell mTOR complex 1 (mTORC1) activity and impairs cytotoxicity. In several reported studies, systemic buffering of tumor acidity enhanced the efficacy of immune checkpoint inhibitors. Paradoxically, we found in a MYC–driven hepatocellular carcinoma model that systemic buffering increased tumor mTORC1 activity, negating inhibition of tumor growth by anti-PD1 treatment. Therefore, in this proof-of-concept study, we tested the metabolic engineering of immune effector cells to mitigate the inhibitory effect of tumor acidity while avoiding side effects associated with systemic buffering. We first overexpressed an activated RHEB in the human NK cell line NK-92, thereby rescuing acid-blunted mTORC1 activity and enhancing cytolytic activity. Then, to directly mitigate the effect of acidity, we ectopically expressed acid extruder proteins. Whereas ectopic expression of carbonic anhydrase IX (CA9) moderately increased mTORC1 activity, it did not enhance effector function. In contrast, overexpressing a constitutively active Na+/H+-exchanger 1 (NHE1; SLC9A1) in NK-92 did not elevate mTORC1 but enhanced degranulation, target engagement, in vitro cytotoxicity, and in vivo antitumor activity. Our findings suggest the feasibility of overcoming the inhibitory effect of the TME by metabolically engineering immune effector cells, which can enhance ACT for better efficacy against solid tumors. Significance: This study demonstrates the feasibility of metabolic engineering immune effector cells to overcome inhibition in the TME, an approach that could enhance the efficacy of adoptive transfer immunotherapy.

Adoptive cell transfer (ACT) immunotherapy has remarkable efficacy against some hematologic malignancies. However, its efficacy in solid tumors is limited by the adverse tumor microenvironment (TME) conditions, most notably that acidity inhibits T and natural killer (NK) cell mTOR complex 1 (mTORC1) activity and impairs cytotoxicity. In several reported studies, systemic buffering of tumor acidity enhanced the efficacy of immune checkpoint inhibitors. Paradoxically, we found in a MYC–driven hepatocellular carcinoma model that systemic buffering increased tumor mTORC1 activity, negating inhibition of tumor growth by anti-PD1 treatment. Therefore, in this proof-of-concept study, we tested the metabolic engineering of immune effector cells to mitigate the inhibitory effect of tumor acidity while avoiding side effects associated with systemic buffering. We first overexpressed an activated RHEB in the human NK cell line NK-92, thereby rescuing acid-blunted mTORC1 activity and enhancing cytolytic activity. Then, to directly mitigate the effect of acidity, we ectopically expressed acid extruder proteins. Whereas ectopic expression of carbonic anhydrase IX (CA9) moderately increased mTORC1 activity, it did not enhance effector function. In contrast, overexpressing a constitutively active Na+/H+-exchanger 1 (NHE1; SLC9A1) in NK-92 did not elevate mTORC1 but enhanced degranulation, target engagement, in vitro cytotoxicity, and in vivo antitumor activity. Our findings suggest the feasibility of overcoming the inhibitory effect of the TME by metabolically engineering immune effector cells, which can enhance ACT for better efficacy against solid tumors.Significance:This study demonstrates the feasibility of metabolic engineering immune effector cells to overcome inhibition in the TME, an approach that could enhance the efficacy of adoptive transfer immunotherapy.

Supplementary Fig. S1. CA9 fails to enhance the cytolytic activity of NK-92 cells in vitro. Supplementary Fig. S2. NHE1 enhances in vitro cytotoxicity of NK-92 cells without affecting proliferation or survival. Supplementary Fig. S3. Representative three-dimensional (3D) reconstruction of confocal microscopic images of antibody-activated NK-92. Supplementary Fig. S4. NHE1 increases the protein expression of c-Myc in NK-92 cells. Supplementary Fig. S5. NHE1 enhances in vitro cytotoxicity of NK-92MI. Supplementary Table S1. Nucleotide sequence of codon-optimized, constitutively active human NHE1 cDNA. Supplementary Table S2. Differentially expressed genes between Na+/H+-exchanger 1 (NHE1)-expressing and empty vector NK-92 cells, ranked by log2(fold change).

Solid tumors possess heterogeneous metabolic microenvironments where oxygen and nutrient availability are plentiful (fertile regions) or scarce (arid regions). While cancer cells residing in fertile regions proliferate rapidly, most cancer cells in vivo reside in arid regions and exhibit a slow-cycling state that renders them chemoresistant. Here, we developed an in vitro system enabling systematic comparison between these populations via transcriptome analysis, metabolomic profiling, and whole-genome CRISPR screening. Metabolic deprivation led to pronounced transcriptional and metabolic reprogramming, resulting in decreased anabolic activities and distinct vulnerabilities. Reductions in anabolic, energy-consuming activities, particularly cell proliferation, were not simply byproducts of the metabolic challenge, but rather essential adaptations. Mechanistically, Bcl-xL played a central role in the adaptation to nutrient and oxygen deprivation. In this setting, Bcl-xL protected quiescent cells from the lethal effects of cell-cycle entry in the absence of adequate nutrients. Moreover, inhibition of Bcl-xL combined with traditional chemotherapy had a synergistic antitumor effect that targeted cycling cells. Bcl-xL expression was strongly associated with poor patient survival despite being confined to the slow-cycling fraction of human pancreatic cancer cells. These findings provide a rationale for combining traditional cancer therapies that target rapidly cycling cells with those that target quiescent, chemoresistant cells associated with nutrient and oxygen deprivation. Significance: The majority of pancreatic cancer cells inhabit nutrient- and oxygen-poor tumor regions and require Bcl-xL for their survival, providing a compelling antitumor metabolic strategy.

The MYC oncogene broadly promotes transcription mediated by all nuclear RNA polymerases, thereby acting as a positive modifier of global gene expression. Here, we report that MYC stimulates the transcription of DANCR, a long noncoding RNA (lncRNA) that is widely overexpressed in human cancer. We identified DANCR through its overexpression in a transgenic model of MYC-induced lymphoma, but found that it was broadly upregulated in many human cancer cell lines and cancers, including most notably in prostate and ovarian cancers. Mechanistic investigations indicated that DANCR limited the expression of cell-cycle inhibitor p21 (CDKN1A) and that the inhibitory effects of DANCR loss on cell proliferation could be partially rescued by p21 silencing. In a xenograft model of human ovarian cancer, a nanoparticle-mediated siRNA strategy to target DANCR in vivo was sufficient to strongly inhibit tumor growth. Our observations expand knowledge of how MYC drives cancer cell proliferation by identifying DANCR as a critical lncRNA widely overexpressed in human cancers.Significance: These findings expand knowledge of how MYC drives cancer cell proliferation by identifying an oncogenic long noncoding RNA that is widely overexpressed in human cancers. Cancer Res; 78(1); 64–74. ©2017 AACR.

Solid tumors possess heterogeneous metabolic microenvironments where oxygen and nutrient availability are plentiful (fertile regions) or scarce (arid regions). While cancer cells residing in fertile regions proliferate rapidly, most cancer cells in vivo reside in arid regions and exhibit a slow-cycling state that renders them chemoresistant. Here, we developed an in vitro system enabling systematic comparison between these populations via transcriptome analysis, metabolomic profiling, and whole-genome CRISPR screening. Metabolic deprivation led to pronounced transcriptional and metabolic reprogramming, resulting in decreased anabolic activities and distinct vulnerabilities. Reductions in anabolic, energy-consuming activities, particularly cell proliferation, were not simply byproducts of the metabolic challenge, but rather essential adaptations. Mechanistically, Bcl-xL played a central role in the adaptation to nutrient and oxygen deprivation. In this setting, Bcl-xL protected quiescent cells from the lethal effects of cell-cycle entry in the absence of adequate nutrients. Moreover, inhibition of Bcl-xL combined with traditional chemotherapy had a synergistic antitumor effect that targeted cycling cells. Bcl-xL expression was strongly associated with poor patient survival despite being confined to the slow-cycling fraction of human pancreatic cancer cells. These findings provide a rationale for combining traditional cancer therapies that target rapidly cycling cells with those that target quiescent, chemoresistant cells associated with nutrient and oxygen deprivation.Significance:The majority of pancreatic cancer cells inhabit nutrient- and oxygen-poor tumor regions and require Bcl-xL for their survival, providing a compelling antitumor metabolic strategy.

Supplementary Table from Bcl-xL Enforces a Slow-Cycling State Necessary for Survival in the Nutrient-Deprived Microenvironment of Pancreatic Cancer

SF 1. Heat map showing the tumor growth inhibition (TGI) of six metabolic inhibitors in PDX panel. SF 2. Tumor growth curves of PDXs treated with phenformin. SF 3. Tumor doubling time of 12 individual PDXs. SF 4. Aggregate tumor doubling time of PDXs treated with vehicle or phenformin.

Supplementary methods and figures S1-S8. (S1): Glutamine dependency and induction of apoptosis on glutamine withdrawal in breast cancer cells. (S2): Cytotoxicity of AOA in breast cancer cells. (S3): Myc regulation of transaminases and glutamine dependency in mouse tumor cell lines. (S4): NMR spectra of AOA-treated compared to vehicle-treated SUM159 cells. (S5): Cell cycle arrest and BrdU incorporation after AOA treatment in SUM149 cells; irreversible growth arrest by AOA in SUM149 and 159 cells; partial rescue of cell death due to glutamine withdrawal by exogenous aspartate in breast cancer cell lines. (S6): Western blot analysis showing change in ER stress markers, energy metabolism and apoptosis by AOA treatment in SUM149, SUM159 and MDA-MB-231 cells. (S7): Tumor growth inhibition by AOA in MCF-7, SUM149 and SUM159 xenografts; immunohistochemistry analysis of cleaved caspase 3 in AOA treated tumors; quantitation of ER stress markers in AOA treated tumors. (S8): Effect of AOA in combination with paclitaxel or carboplatin in SUM149 and SUM159 xenografts; up regulation of c-myc in carboplatin and paclitaxel treated MDA-MB-231 xenografts.

Supplementary Data from Bcl-xL Enforces a Slow-Cycling State Necessary for Survival in the Nutrient-Deprived Microenvironment of Pancreatic Cancer

Table S1. MYC-regulated genes in P493-6 cell line Table S1 provides expression profiles of genes in response to MYC induction in the lymphoma model cell line P493-6.

Supplementary Figure from Bcl-xL Enforces a Slow-Cycling State Necessary for Survival in the Nutrient-Deprived Microenvironment of Pancreatic Cancer

Purpose: Glutamine addiction in c-MYC–overexpressing breast cancer is targeted by the aminotransferase inhibitor, aminooxyacetate (AOA). However, the mechanism of ensuing cell death remains unresolved.Experimental Design: A correlation between glutamine dependence for growth and c-MYC expression was studied in breast cancer cell lines. The cytotoxic effects of AOA, its correlation with high c-MYC expression, and effects on enzymes in the glutaminolytic pathway were investigated. AOA-induced cell death was assessed by measuring changes in metabolite levels by magnetic resonance spectroscopy (MRS), the effects of amino acid depletion on nucleotide synthesis by cell-cycle and bromodeoxyuridine (BrdUrd) uptake analysis, and activation of the endoplasmic reticulum (ER) stress–mediated pathway. Antitumor effects of AOA with or without common chemotherapies were determined in breast cancer xenografts in immunodeficient mice and in a transgenic MMTV-rTtA-TetO-myc mouse mammary tumor model.Results: We established a direct correlation between c-MYC overexpression, suppression of glutaminolysis, and AOA sensitivity in most breast cancer cells. MRS, cell-cycle analysis, and BrdUrd uptake measurements indicated depletion of aspartic acid and alanine leading to cell-cycle arrest at S-phase by AOA. Activation of components of the ER stress–mediated pathway, initiated through GRP78, led to apoptotic cell death. AOA inhibited growth of SUM159, SUM149, and MCF-7 xenografts and c-myc–overexpressing transgenic mouse mammary tumors. In MDA-MB-231, AOA was effective only in combination with chemotherapy.Conclusions: AOA mediates its cytotoxic effects largely through the stress response pathway. The preclinical data of AOA's effectiveness provide a strong rationale for further clinical development, particularly for c-MYC–overexpressing breast cancers. Clin Cancer Res; 21(14); 3263–73. ©2015 AACR.

Figure S1. The expression of DANCR is upregulated by MYC activation in P493-6 cells Normalized expression of DANCR (KIAA0114) in MYC-OFF or MYC-ON P493-6 cells measured by exon microarrays (PLoS One 6, e26057). Three independent array experiments were performed. Figure S2. Human-mouse sequence alignment analysis of DANCR and mDancr Figure S3. The mRNA expression of the DANCR adjacent protein-coding genes in cells transduced with DANCR-specific siRNA Control and DANCR siRNAs were transduced into PC3 cells. After 48 and 72 hours, the mRNA expression levels of the DANCR adjacent protein-coding genes (ERVMER34-1 and UPS46) were analyzed by qRT-PCR. Figure S4. Effects of two DANCR targeting antisense morpholinos on P493-6 cell proliferation and p21 expression. A. RT-PCR analysis of DANCR expression in P493-6 after being treated by various concentrations of the two DANCR antisense morpholinos. B. Effects of two different morpholinos targeting DANCR on proliferation of P493-6 cells as compared with control morpholino. C. Induction of P21 by loss of DANCR expression in P493-6 cells treated by two concentrations of the two DANCR antisense morpholinos.

Table S1. MYC-regulated genes in P493-6 cell line Table S2. MYC-regulated lncRNAs in P493-6 cell line Table S3. MYC-regulated lncRNAs in CCLE Table S4. GO analysis of genes that affected by DANCR knockdown in PC3 cells Table S5. Top 500 up-regulated and down-regulated genes by knockdown of DANCR in PC3 cells. Figure S1. The expression of DANCR is upregulated by MYC activation in P493-6 cells Figure S2. Human-mouse sequence alignment analysis of DANCR and mDancr Figure S3. The mRNA expression of the DANCR adjacent protein-coding genes in cells transduced with DANCR-specific siRNA Figure S4. Effects of two DANCR targeting antisense morpholinos on P493-6 cell proliferation and p21 expression.

Purpose: To identify effective metabolic inhibitors to suppress the aggressive growth of pancreatic ductal adenocarcinoma (PDAC), we explored the in vivo antitumor efficacy of metabolic inhibitors, as single agents, in a panel of patient-derived PDAC xenograft models (PDX) and investigated whether genomic alterations of tumors correlate with the sensitivity to metabolic inhibitors.Experimental Design: Mice with established PDAC tumors from 6 to 13 individual PDXs were randomized and treated, once daily for 4 weeks, with either sterile PBS (vehicle) or the glutaminase inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), transaminase inhibitor aminooxyacetate (AOA), pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA), autophagy inhibitor chloroquine (CQ), and mitochondrial complex I inhibitor phenformin/metformin.Results: Among the agents tested, phenformin showed significant tumor growth inhibition (>30% compared with vehicle) in 5 of 12 individual PDXs. Metformin, at a fivefold higher dose, displayed significant tumor growth inhibition in 3 of 12 PDXs similar to BPTES (2/8 PDXs) and DCA (2/6 PDXs). AOA and CQ had the lowest response rates. Gene set enrichment analysis conducted using the baseline gene expression profile of pancreatic tumors identified a gene expression signature that inversely correlated with phenformin sensitivity, which is in agreement with the phenformin gene expression signature of NIH Library of Integrated Network-based Cellular Signatures (LINCS). The PDXs that were more sensitive to phenformin showed a baseline reduction in amino acids and elevation in oxidized glutathione. There was no correlation between phenformin response and genetic alterations in KRAS, TP53, SMAD4, or PTEN.Conclusions: Phenformin treatment showed relatively higher antitumor efficacy against established PDAC tumors, compared with the efficacy of other metabolic inhibitors and metformin. Phenformin treatment significantly diminished PDAC tumor progression and prolonged tumor doubling time. Overall, our results serve as a foundation for further evaluation of phenformin as a therapeutic agent in pancreatic cancer. Clin Cancer Res; 23(18); 5639–47. ©2017 AACR.

PDF file - 116K, Structure of the MCT1 inhibitors AR-C122982 (SR13800) and AR-C155858 (SR13801). Cell cycle analysis, viability, clonogenecity and lactate transport in the indicated cells treated with SR13800. Proliferation of MCF7 cells overexpressing MCT1 or MCT4.

PDF file - 358K

Supplementary Figure 2 from Inhibition of Glutaminase Preferentially Slows Growth of Glioma Cells with Mutant IDH1

PDF file - 728K, Immunohistochemistry analyses of MCT1 and MCT4, and survival and tumor analyses of Raji lymphoma and T47D breast cancer cell xenografts.

Supplementary Figure Legends 1-7 from Inhibition of Glutaminase Preferentially Slows Growth of Glioma Cells with Mutant IDH1

Supplementary Methods from Inhibition of Glutaminase Preferentially Slows Growth of Glioma Cells with Mutant IDH1

PDF file - 78K, Effects of combined treatment of SR13800 and metformin on intracellular lactate and proliferation of Raji BL cells, and upon the proliferation of MCT1-expressing lymphoma and breast cancer cells.