Metabolic engineering against the arginine microenvironment enhances CAR-T cell proliferation and therapeutic activity.

Hematological and solid cancers catabolize the semiessential amino acid arginine to drive cell proliferation. However, the resulting low arginine microenvironment also impairs chimeric antigen receptor T cells (CAR-T) cell proliferation, limiting their efficacy in clinical trials against hematological and solid malignancies. T cells are susceptible to the low arginine microenvironment because of the low expression of the arginine resynthesis enzymes argininosuccinate synthase (ASS) and ornithine transcarbamylase (OTC). We demonstrate that T cells can be reengineered to express functional ASS or OTC enzymes, in concert with different chimeric antigen receptors. Enzyme modifications increase CAR-T cell proliferation, with no loss of CAR cytotoxicity or increased exhaustion. In vivo, enzyme-modified CAR-T cells lead to enhanced clearance of leukemia or solid tumor burden, providing the first metabolic modification to enhance CAR-T cell therapies.

eIF4EBP3L acts as a gatekeeper of TORC1 in activity-dependent muscle growth by specifically regulating Mef2ca translational initiation.

Muscle fiber size is activity-dependent and clinically important in ageing, bed-rest, and cachexia, where muscle weakening leads to disability, prolonged recovery times, and increased costs. Inactivity causes muscle wasting by triggering protein degradation and may simultaneously prevent protein synthesis. During development, muscle tissue grows by several mechanisms, including hypertrophy of existing fibers. As in other tissues, the TOR pathway plays a key role in promoting muscle protein synthesis by inhibition of eIF4EBPs (eukaryotic Initiation Factor 4E Binding Proteins), regulators of the translational initiation. Here, we tested the role of TOR-eIF4EBP in a novel zebrafish muscle inactivity model. Inactivity triggered up-regulation of eIF4EBP3L (a zebrafish homolog of eIF4EBP3) and diminished myosin and actin content, myofibrilogenesis, and fiber growth. The changes were accompanied by preferential reduction of the muscle transcription factor Mef2c, relative to Myod and Vinculin. Polysomal fractionation showed that Mef2c decrease was due to reduced translation of mef2ca mRNA. Loss of Mef2ca function reduced normal muscle growth and diminished the reduction in growth caused by inactivity. We identify eIF4EBP3L as a key regulator of Mef2c translation and protein level following inactivity; blocking eIF4EBP3L function increased Mef2ca translation. Such blockade also prevented the decline in mef2ca translation and level of Mef2c and slow myosin heavy chain proteins caused by inactivity. Conversely, overexpression of active eIF4EBP3L mimicked inactivity by decreasing the proportion of mef2ca mRNA in polysomes, the levels of Mef2c and slow myosin heavy chain, and myofibril content. Inhibiting the TOR pathway without the increase in eIF4EBP3L had a lesser effect on myofibrilogenesis and muscle size. These findings identify eIF4EBP3L as a key TOR-dependent regulator of muscle fiber size in response to activity. We suggest that by selectively inhibiting translational initiation of mef2ca and other mRNAs, eIF4EBP3L reprograms the translational profile of muscle, enabling it to adjust to new environmental conditions.

Metalloprotease type III effectors that specifically cleave JNK and NF-κB.

Two major arms of the inflammatory response are the NF-κB and c-Jun N-terminal kinase (JNK) pathways. Here, we show that enteropathogenic Escherichia coli (EPEC) employs the type III secretion system to target these two signalling arms by injecting host cells with two effector proteins, NleC and NleD. We provide evidence that NleC and NleD are Zn-dependent endopeptidases that specifically clip and inactivate RelA (p65) and JNK, respectively, thus blocking NF-κB and AP-1 activation. We show that NleC and NleD co-operate and complement other EPEC effectors in accomplishing maximal inhibition of IL-8 secretion. This is a remarkable example of a pathogen using multiple effectors to manipulate systematically the host inflammatory response signalling network.

Fumarase: a mitochondrial metabolic enzyme and a cytosolic/nuclear component of the DNA damage response.

In eukaryotes, fumarase (FH in human) is a well-known tricarboxylic-acid-cycle enzyme in the mitochondrial matrix. However, conserved from yeast to humans is a cytosolic isoenzyme of fumarase whose function in this compartment remains obscure. A few years ago, FH was surprisingly shown to underlie a tumor susceptibility syndrome, Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). A biallelic inactivation of FH has been detected in almost all HLRCC tumors, and therefore FH was suggested to function as a tumor suppressor. Recently it was suggested that FH inhibition leads to elevated intracellular fumarate, which in turn acts as a competitive inhibitor of HPH (HIF prolyl hydroxylase), thereby causing stabilization of HIF (Hypoxia-inducible factor) by preventing proteasomal degradation. The transcription factor HIF increases the expression of angiogenesis regulated genes, such as VEGF, which can lead to high microvessel density and tumorigenesis. Yet this mechanism does not fully explain the large cytosolic population of fumarase molecules. We constructed a yeast strain in which fumarase is localized exclusively to mitochondria. This led to the discovery that the yeast cytosolic fumarase plays a key role in the protection of cells from DNA damage, particularly from DNA double-strand breaks. We show that the cytosolic fumarase is a member of the DNA damage response that is recruited from the cytosol to the nucleus upon DNA damage induction. This function of fumarase depends on its enzymatic activity, and its absence in cells can be complemented by high concentrations of fumaric acid. Our findings suggest that fumarase and fumaric acid are critical elements of the DNA damage response, which underlies the tumor suppressor role of fumarase in human cells and which is most probably HIF independent. This study shows an exciting crosstalk between primary metabolism and the DNA damage response, thereby providing a scenario for metabolic control of tumor propagation.

Establishment of In Vivo Acquired Resistance to Chemotherapy Via Individual Dose Escalation Treatment Regime.

The vast majority of cancer deaths are the result of drug resistance. The lack of superior preclinical models that better reflect the complexity of relapsed disease hinders the development of novel therapeutics. 2D and 3D in vitro cell-based assays have provided some information, but this is limited and does not consider the role of the tumor microenvironment. The development of an in vivo assay can allow to generate resistance, while taking into account the role of the tumor microenvironment and the tumor structure. To achieve this, we have developed an in vivo dose-escalation protocol that models the acquisition of resistance. This model of chemo-resistant neuroblastoma presented with metastases and a genetic signature characteristic of clinical relapsed tumors (Yogev et al. Cancer Res. 79:5382-5393, 2019). We believe that this protocol can be used to generate faithful models for other types of relapse disease; these could serve as reliable tools while developing novel therapies.

Indisulam targets RNA splicing and metabolism to serve as a therapeutic strategy for high-risk neuroblastoma.

Neuroblastoma is the most common paediatric solid tumour and prognosis remains poor for high-risk cases despite the use of multimodal treatment. Analysis of public drug sensitivity data showed neuroblastoma lines to be sensitive to indisulam, a molecular glue that selectively targets RNA splicing factor RBM39 for proteosomal degradation via DCAF15-E3-ubiquitin ligase. In neuroblastoma models, indisulam induces rapid loss of RBM39, accumulation of splicing errors and growth inhibition in a DCAF15-dependent manner. Integrative analysis of RNAseq and proteomics data highlight a distinct disruption to cell cycle and metabolism. Metabolic profiling demonstrates metabolome perturbations and mitochondrial dysfunction resulting from indisulam. Complete tumour regression without relapse was observed in both xenograft and the Th-MYCN transgenic model of neuroblastoma after indisulam treatment, with RBM39 loss, RNA splicing and metabolic changes confirmed in vivo. Our data show that dual-targeting of metabolism and RNA splicing with anticancer indisulam is a promising therapeutic approach for high-risk neuroblastoma.

In Vivo Modeling of Chemoresistant Neuroblastoma Provides New Insights into Chemorefractory Disease and Metastasis.

Neuroblastoma is a pediatric cancer that is frequently metastatic and resistant to conventional treatment. In part, a lack of natively metastatic, chemoresistant in vivo models has limited our insight into the development of aggressive disease. The Th-MYCN genetically engineered mouse model develops rapidly progressive chemosensitive neuroblastoma and lacks clinically relevant metastases. To study tumor progression in a context more reflective of clinical therapy, we delivered multicycle treatment with cyclophosphamide to Th-MYCN mice, individualizing therapy using MRI, to generate the Th-MYCN CPM32 model. These mice developed chemoresistance and spontaneous bone marrow metastases. Tumors exhibited an altered immune microenvironment with increased stroma and tumor-associated fibroblasts. Analysis of copy number aberrations revealed genomic changes characteristic of human MYCN-amplified neuroblastoma, specifically copy number gains at mouse chromosome 11, syntenic with gains on human chromosome 17q. RNA sequencing revealed enriched expression of genes associated with 17q gain and upregulation of genes associated with high-risk neuroblastoma, such as the cell-cycle regulator cyclin B1-interacting protein 1 (Ccnb1ip1) and thymidine kinase (TK1). The antiapoptotic, prometastatic JAK-STAT3 pathway was activated in chemoresistant tumors, and treatment with the JAK1/JAK2 inhibitor CYT387 reduced progression of chemoresistant tumors and increased survival. Our results highlight that under treatment conditions that mimic chemotherapy in human patients, Th-MYCN mice develop genomic, microenvironmental, and clinical features reminiscent of human chemorefractory disease. The Th-MYCN CPM32 model therefore is a useful tool to dissect in detail mechanisms that drive metastasis and chemoresistance, and highlights dysregulation of signaling pathways such as JAK-STAT3 that could be targeted to improve treatment of aggressive disease. SIGNIFICANCE: An in vivo mouse model of high-risk treatment-resistant neuroblastoma exhibits changes in the tumor microenvironment, widespread metastases, and sensitivity to JAK1/2 inhibition.

Macrophage-Derived IL1ß and TNFα Regulate Arginine Metabolism in Neuroblastoma.

Neuroblastoma is the most common childhood solid tumor, yet the prognosis for high-risk disease remains poor. We demonstrate here that arginase 2 (ARG2) drives neuroblastoma cell proliferation via regulation of arginine metabolism. Targeting arginine metabolism, either by blocking cationic amino acid transporter 1 (CAT-1)-dependent arginine uptake in vitro or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival. Tumor cells polarized infiltrating monocytes to an M1-macrophage phenotype, which released IL1ß and TNFα in a RAC-alpha serine/threonine-protein kinase (AKT)-dependent manner. IL1ß and TNFα established a feedback loop to upregulate ARG2 expression via p38 and extracellular regulated kinases 1/2 (ERK1/2) signaling in neuroblastoma and neural crest-derived cells. Proteomic analysis revealed that enrichment of IL1ß and TNFα in stage IV human tumor microenvironments was associated with a worse prognosis. These data thus describe an immune-metabolic regulatory loop between tumor cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited. SIGNIFICANCE: These findings illustrate that cross-talk between myeloid cells and tumor cells creates a metabolic regulatory loop that promotes neuroblastoma progression.

p53 Loss in MYC-Driven Neuroblastoma Leads to Metabolic Adaptations Supporting Radioresistance.

Neuroblastoma is the most common childhood extracranial solid tumor. In high-risk cases, many of which are characterized by amplification of MYCN, outcome remains poor. Mutations in the p53 (TP53) tumor suppressor are rare at diagnosis, but evidence suggests that p53 function is often impaired in relapsed, treatment-resistant disease. To address the role of p53 loss of function in the development and pathogenesis of high-risk neuroblastoma, we generated a MYCN-driven genetically engineered mouse model in which the tamoxifen-inducible p53ER(TAM) fusion protein was expressed from a knock-in allele (Th-MYCN/Trp53(KI)). We observed no significant differences in tumor-free survival between Th-MYCN mice heterozygous for Trp53(KI) (n = 188) and Th-MYCN mice with wild-type p53 (n = 101). Conversely, the survival of Th-MYCN/Trp53(KI/KI) mice lacking functional p53 (n = 60) was greatly reduced. We found that Th-MYCN/Trp53(KI/KI) tumors were resistant to ionizing radiation (IR), as expected. However, restoration of functional p53ER(TAM) reinstated sensitivity to IR in only 50% of Th-MYCN/Trp53(KI/KI) tumors, indicating the acquisition of additional resistance mechanisms. Gene expression and metabolic analyses indicated that the principal acquired mechanism of resistance to IR in the absence of functional p53 was metabolic adaptation in response to chronic oxidative stress. Tumors exhibited increased antioxidant metabolites and upregulation of glutathione S-transferase pathway genes, including Gstp1 and Gstz1, which are associated with poor outcome in human neuroblastoma. Accordingly, glutathione depletion by buthionine sulfoximine together with restoration of p53 activity resensitized tumors to IR. Our findings highlight the complex pathways operating in relapsed neuroblastomas and the need for combination therapies that target the diverse resistance mechanisms at play. Cancer Res; 76(10); 3025-35. ©2016 AACR.

Jun proteins inhibit autophagy and induce cell death.

Starvation induces a vigorous autophagic response to enhance cellular survival, whereas nutrient and serum supplementation inhibit autophagy and induce an intensive transcriptional burst that enables cellular proliferation. We recently found that some of the genes induced by serum and growth factors--the immediate early proteins JunB and c-Jun--inhibit autophagy. Deregulation of JunB expression when autophagy is specifically required, tilts the fate of starved cells to apoptosis.

Jun proteins are starvation-regulated inhibitors of autophagy.

The growing number of biological functions affected by autophagy ascribes a special significance to identification of factors regulating it. The activator protein-1 (AP-1) transcription factors are involved in most aspects of cellular proliferation, death, or survival, yet no information regarding their involvement in autophagy is available. Here, we show that the AP-1 proteins JunB and c-Jun, but not JunD, c-Fos, or Fra-1, inhibit autophagy. JunB inhibits autophagy induced by starvation, overexpression of a short form of ARF (smARF), a potent inducer of autophagy, or even after rapamycin treatment. In agreement, acute repression of JunB expression, by JunB knockdown, potently induces autophagy. As expected from autophagy-inhibiting proteins, Jun B and c-Jun expression is reduced by starvation. Decrease in JunB mRNA expression and posttranscriptional events downregulate JunB protein expression after starvation. The inhibition of autophagy by JunB is not mediated by mammalian target of rapamycin (mTOR) regulation, as it occurs also in the absence of mTOR activity, and autophagy induced by JunB knockdown is not correlated with changes in mTOR activity. Nevertheless, the transcriptional activities of c-Jun and JunB are required for autophagy inhibition, and JunB incapable of heterodimerizing is a less effective inhibitor of autophagy. Most importantly, inhibition of autophagy in starved HeLa cells by JunB enhances apoptotic cell death. We suggest that JunB and c-Jun are regulators of autophagy whose expression responds to autophagy-inducing signals.

DNA damage-dependent translocation of B23 and p19 ARF is regulated by the Jun N-terminal kinase pathway.

The dynamic behavior of the nucleolus plays a role in the detection of and response to DNA damage of cells. Two nucleolar proteins, p14(ARF)/p19(ARF) and B23, were shown to translocate out of the nucleolus after exposure of cells to DNA-damaging agents. This translocation affects multiple cellular functions, such as DNA repair, proliferation, and survival. In this study, we identify a pathway and scrutinize the mechanisms leading to the translocation of these proteins after exposure of cells to DNA-damaging agents. We show that redistribution of B23 and p19(ARF) after the exposure to genotoxic stress occurs preferentially when the c-Jun-NH(2)-kinase (JNK) pathway is activated and is inhibited when the JNK pathway is impaired. The stress-induced translocation of alternative reading frame (ARF) is JNK dependent and mediated by two activator proteins, c-Jun and JunB. Thr(91) and Thr(93) of c-Jun are required for the translocation, but the transcriptional activity of c-Jun is dispensable. Instead, c-Jun interacts with B23 in a dose-dependent manner. c-Jun itself is excluded from the nucleolus in a JNK-dependent manner. Hence, we suggest that c-Jun translocates B23 and ARF from the nucleolus after JNK activation by means of protein interactions. In senescent cells, JNK activity and c-Jun levels are reduced concomitantly with ARF nucleolar accumulation, and UV radiation does not cause the translocation of ARF.

Induction of transcriptionally active Jun proteins regulates drug-induced senescence.

The drug hydroxyurea (HU) is used for cancer therapy and treatment of sickle cell anemia. It inhibits cell cycle progression by blocking DNA synthesis and drives cells to undergo apoptosis or enter senescence. We demonstrate here that HU induces the expression of two AP-1 proteins, c-Jun and JunB, which exert antagonistic effects on the cell cycle. Moreover, the induction of c-Jun is observed following treatment with two other drugs that inhibit the cell cycle in S phase, aphidicolin and camptothecin. The induction of c-Jun, which promotes cell cycle progression, up-regulates expression of cyclin D after exposure of cells to HU. Deficiency in c-jun prevents elevation of cyclin D expression and extends entrance into HU-induced senescence but also renders cells more resistant to HU-dependent apoptosis. The induction of c-Jun is independent of JNK activity, and additionally, of c-Jun autoregulatory activity but is inhibited upon inhibition of protein kinase C activity. Therefore, we suggest that c-Jun activity prevents drug-induced senescence. Conversely, the JunB target gene, tumor suppressor p16(INK4a), a cyclin-dependent kinase inhibitor essential for the induction of drug-induced senescence, is also up-regulated by HU in a JunB-dependent manner. Constitutive expression of JunB up-regulates p16(INK4a) and increases the sensitivity of mouse fibroblasts to drug-induced-senescence. Thus, we suggest that in contrast to c-Jun, JunB drives cells to enter HU-dependent senescence. The effect of HU treatment, which regulates the intricate web of AP-1 transcription, depends on the balance between c-Jun and JunB activities.