Dual inhibition of IDO1/TDO2 enhances anti-tumor immunity in platinum-resistant non-small cell lung cancer.

BACKGROUND: The impact of non-small cell lung cancer (NSCLC) metabolism on the immune microenvironment is not well understood within platinum resistance. We have identified crucial metabolic differences between cisplatin-resistant (CR) and cisplatin-sensitive (CS) NSCLC cells with elevated indoleamine 2,3-dioxygenase-1 (IDO1) activity in CR, recognized by increased kynurenine (KYN) production. METHODS: Co-culture, syngeneic, and humanize mice models were utilized. C57BL/6 mice were inoculated with either Lewis lung carcinoma mouse cells (LLC) or their platinum-resistant counterpart (LLC-CR) cells. Humanized mice were inoculated with either A (human CS cells) or ALC (human CR cells). Mice were treated with either IDO1 inhibitor or TDO2 (tryptophan 2,3-dioxygenase-2) inhibitor at 200 mg/kg P.O. once a day for 15 days; or with a new-in-class, IDO1/TDO2 dual inhibitor AT-0174 at 170 mg/kg P.O. once a day for 15 days with and without anti-PD1 antibody (10 mg/kg, every 3 days). Immune profiles and KYN and tryptophan (TRP) production were evaluated. RESULTS: CR tumors exhibited a more highly immunosuppressive environment that debilitated robust anti-tumor immune responses. IDO1-mediated KYN production from CR cells suppressed NKG2D on immune effector natural killer (NK) and CD8+ T cells and enhanced immunosuppressive populations of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Importantly, while selective IDO1 inhibition attenuated CR tumor growth, it concomitantly upregulated the TDO2 enzyme. To overcome the compensatory induction of TDO2 activity, we employed the IDO1/TDO2 dual inhibitor, AT-0174. Dual inhibition of IDO1/TDO2 in CR mice suppressed tumor growth to a greater degree than IDO1 inhibition alone. Significant enhancement in NKG2D frequency on NK and CD8+ T cells and a reduction in Tregs and MDSCs were observed following AT-1074 treatment. PD-L1 (programmed death-ligand-1) expression was increased in CR cells; therefore, we assessed dual inhibition + PD1 (programmed cell death protein-1) blocking and report profound anti-tumor growth and improved immunity in CR tumors which in turn extended overall survival in mice. CONCLUSION: Our study reports the presence of platinum-resistant lung tumors that utilize both IDO1/TDO2 enzymes for survival, and to escape immune surveillance as a consequence of KYN metabolites. We also report early in vivo data in support of the potential therapeutic efficacy of the dual IDO1/TDO2 inhibitor AT-0174 as a part of immuno-therapeutic treatment that disrupts tumor metabolism and enhances anti-tumor immunity.

Enhancing the Effect of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Signaling and Arginine Deprivation in Melanoma.

Melanoma as a very aggressive type of cancer is still in urgent need of improved treatment. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and arginine deiminase (ADI-PEG20) are two of many suggested drugs for treating melanoma. Both have shown anti-tumor activities without harming normal cells. However, resistance to both drugs has also been noted. Studies on the mechanism of action of and resistance to these drugs provide multiple targets that can be utilized to increase the efficacy and overcome the resistance. As a result, combination strategies have been proposed for these drug candidates with various other agents, and achieved enhanced or synergistic anti-tumor effect. The combination of TRAIL and ADI-PEG20 as one example can greatly enhance the cytotoxicity to melanoma cells including those resistant to the single component of this combination. It is found that combination treatment generally can alter the expression of the components of cell signaling in melanoma cells to favor cell death. In this paper, the signaling of TRAIL and ADI-PEG20-induced arginine deprivation including the main mechanism of resistance to these drugs and exemplary combination strategies is discussed. Finally, factors hampering the clinical application of both drugs, current and future development to overcome these hurdles are briefly discussed.

Cisplatin Resistance and Redox-Metabolic Vulnerability: A Second Alteration.

The development of drug resistance in tumors is a major obstacle to effective cancer chemotherapy and represents one of the most significant complications to improving long-term patient outcomes. Despite early positive responsiveness to platinum-based chemotherapy, the majority of lung cancer patients develop resistance. The development of a new combination therapy targeting cisplatin-resistant (CR) tumors may mark a major improvement as salvage therapy in these patients. The recent resurgence in research into cellular metabolism has again confirmed that cancer cells utilize aerobic glycolysis ("the Warburg effect") to produce energy. Hence, this observation still remains a characteristic hallmark of altered metabolism in certain cancer cells. However, recent evidence promotes another concept wherein some tumors that acquire resistance to cisplatin undergo further metabolic alterations that increase tumor reliance on oxidative metabolism (OXMET) instead of glycolysis. Our review focuses on molecular changes that occur in tumors due to the relationship between metabolic demands and the importance of NAD+ in redox (ROS) metabolism and the crosstalk between PARP-1 (Poly (ADP ribose) polymerase-1) and SIRTs (sirtuins) in CR tumors. Finally, we discuss a role for the tumor metabolites of the kynurenine pathway (tryptophan catabolism) as effectors of immune cells in the tumor microenvironment during acquisition of resistance in CR cells. Understanding these concepts will form the basis for future targeting of CR cells by exploiting redox-metabolic changes and their consequences on immune cells in the tumor microenvironment as a new approach to improve overall therapeutic outcomes and survival in patients who fail cisplatin.

Targeting the Proline-Glutamine-Asparagine-Arginine Metabolic Axis in Amino Acid Starvation Cancer Therapy.

Proline, glutamine, asparagine, and arginine are conditionally non-essential amino acids that can be produced in our body. However, they are essential for the growth of highly proliferative cells such as cancers. Many cancers express reduced levels of these amino acids and thus require import from the environment. Meanwhile, the biosynthesis of these amino acids is inter-connected but can be intervened individually through the inhibition of key enzymes of the biosynthesis of these amino acids, resulting in amino acid starvation and cell death. Amino acid starvation strategies have been in various stages of clinical applications. Targeting asparagine using asparaginase has been approved for treating acute lymphoblastic leukemia. Targeting glutamine and arginine starvations are in various stages of clinical trials, and targeting proline starvation is in preclinical development. The most important obstacle of these therapies is drug resistance, which is mostly due to reactivation of the key enzymes involved in biosynthesis of the targeted amino acids and reprogramming of compensatory survival pathways via transcriptional, epigenetic, and post-translational mechanisms. Here, we review the interactive regulatory mechanisms that control cellular levels of these amino acids for amino acid starvation therapy and how drug resistance is evolved underlying treatment failure.

Unilateral Fourth Nerve Palsy due to Presumed Metastatic Melanoma.

ABSTRACT: An 81-year-old man with a history of metastatic melanoma presented with sudden onset of painless, binocular vertical diplopia. The clinical examination was consistent with a right fourth nerve palsy. An MRI of the head revealed a mass dorsal to the right tectum at the level of the inferior colliculus. An MRI just 4 months prior did not show a lesion in that location. An MRA of the head did not show an aneurysm. This is a rare case of an isolated fourth nerve palsy believed to be due to metastatic melanoma compressing the nerve along the dorsal midbrain.

Single-cell analysis reveals new evolutionary complexity in uveal melanoma.

Uveal melanoma (UM) is a highly metastatic cancer that, in contrast to cutaneous melanoma, is largely unresponsive to checkpoint immunotherapy. Here, we interrogate the tumor microenvironment at single-cell resolution using scRNA-seq of 59,915 tumor and non-neoplastic cells from 8 primary and 3 metastatic samples. Tumor cells reveal novel subclonal genomic complexity and transcriptional states. Tumor-infiltrating immune cells comprise a previously unrecognized diversity of cell types, including CD8+ T cells predominantly expressing the checkpoint marker LAG3, rather than PD1 or CTLA4. V(D)J analysis shows clonally expanded T cells, indicating that they are capable of mounting an immune response. An indolent liver metastasis from a class 1B UM is infiltrated with clonally expanded plasma cells, indicative of antibody-mediated immunity. This complex ecosystem of tumor and immune cells provides new insights into UM biology, and LAG3 is identified as a potential candidate for immune checkpoint blockade in patients with high risk UM.

Collaboration Between RSK-EphA2 and Gas6-Axl RTK Signaling in Arginine Starvation Response That Confers Resistance to EGFR Inhibitors.

Many human malignancies require extracellular arginine (Arg) for survival because the key enzyme for de novo Arg biosynthesis, argininosuccinate synthetase 1 (ASS1), is silenced. Recombinant arginine deiminase (ADI-PEG20), which digests extracellular Arg, has been in clinical trials for treating ASS1-negative tumors. Reactivation of ASS1 is responsible for the treatment failure. We previously demonstrated that ASS1 reactivation is transcriptionally regulated by c-Myc via the upstream Gas6-Axl tyrosine kinase (RTK) signal. Here, we report that another RTK EphA2 is coactivated via PI3K-ERK/RSK1 pathway in a ligand-independent mechanism. EphA2 is also regulated by c-Myc. Moreover, we found that knockdown Axl upregulates EphA2 expression, demonstrating cross-talk between these RTKs. ADIR cell lines exhibits enhanced sensitivities to nutrient deprivation such as charcoal-stripped FBS and multiple RTK inhibitor foretinib but resistance to EGFR inhibitors. Knockdown EphA2, and to lesser extent, Axl, overcomes EGFRi resistance. c-Myc inhibitor JQ1 can also sensitize ADIR cells to ADI-PEG20. This study elucidates molecular interactions of multiple RTKs in Arg-stress response and offers approaches for developing strategies of overcoming ADI-PEG20 resistance.

Targeting the Kynurenine Pathway for the Treatment of Cisplatin-Resistant Lung Cancer.

Cisplatin resistance is a major barrier in the effective treatment of lung cancer. Cisplatin-resistant (CR) lung cancer cells do not primarily use glucose but rather consume amino acids such as glutamine and tryptophan (Trp) for survival. CR cells activate the kynurenine (KYN) pathway (KP) to cope with excessive reactive oxygen species (ROS) and maintain homeostasis for growth and proliferation. Consequently, indoleamine 2,3-dioxygenase-1 (IDO1) becomes an essential enzyme for CR cells' survival because it initiates and regulates the first step in the KP. Increased IDO1 activities and ROS levels are found in CR cells versus cisplatin-sensitive lung cancer. Importantly, significantly greater KYN/Trp ratio (P = 0.005) is detected in serum of patients who fail cisplatin when compared with naïve treatment. Knocking down IDO1 using shRNA or IDO1 inhibitors heightens ROS levels and results in a significant growth inhibitory effect only on CR cells and not on cisplatin-sensitive cells. Exposing CR cells to antioxidant (TIRON) results in suppression of IDO1 activity and confers resistance to IDO1 inhibition, indicating an interrelationship between ROS and IDO1. Because KYN plays a critical role in reprogramming naïve T cells to the immune-suppressive regulatory T-cell (T-reg) phenotype, we observed higher expression of TGFß, FoxP3, and CD4+CD25+ in mice bearing CR tumors compared with tumors from cisplatin-sensitive counterparts. IMPLICATIONS: Findings suggest that the enzyme-inhibitory activity and antitumor efficacy of IDO1 inhibitors rely in part on ROS levels, arguing that IDO1 expression alone may be insufficient to determine the clinical benefits for this class of experimental cancer drugs. Importantly, IDO1 inhibitors may be more suitable to treat patients with lung cancer who failed cisplatin therapy than naïve treatment patients.

First-in-Human Phase I Study of Fisogatinib (BLU-554) Validates Aberrant FGF19 Signaling as a Driver Event in Hepatocellular Carcinoma.

Outcomes for patients with advanced hepatocellular carcinoma (HCC) remain poor despite recent progress in drug development. Emerging data implicate FGF19 as a potential HCC driver, suggesting its receptor, FGFR4, as a novel therapeutic target. We evaluated fisogatinib (BLU-554), a highly potent and selective oral FGFR4 inhibitor, in a phase I dose-escalation/dose-expansion study in advanced HCC using FGF19 expression measured by IHC as a biomarker for pathway activation. For dose escalation, 25 patients received 140 to 900 mg fisogatinib once daily; the maximum tolerated dose (600 mg once daily) was expanded in 81 patients. Fisogatinib was well tolerated; most adverse events were manageable, grade 1/2 gastrointestinal events, primarily diarrhea, nausea, and vomiting. Across doses, the overall response rate was 17% in FGF19-positive patients [median duration of response: 5.3 months (95% CI, 3.7-not reached)] and 0% in FGF19-negative patients. These results validate FGFR4 as a targetable driver in FGF19-positive advanced HCC. SIGNIFICANCE: Fisogatinib elicited clinical responses in patients with tumor FGF19 overexpression in advanced HCC. These results validate the oncogenic driver role of the FGFR4 pathway in HCC and the use of FGF19 as a biomarker for patient selection.See related commentary by Subbiah and Pal, p. 1646.This article is highlighted in the In This Issue feature, p. 1631.

Phase 2 study of pembrolizumab and circulating biomarkers to predict anticancer response in advanced, unresectable hepatocellular carcinoma.

BACKGROUND: Checkpoint inhibitors have shown modest activity in patients with advanced hepatocellular carcinoma (HCC). Herein, the authors report a prospective single-institution clinical/translational phase 2 study of pembrolizumab in patients with advanced HCC and circulating biomarkers closely related to response. METHODS: Pembrolizumab was administered at a dose of 200 mg intravenously every 3 weeks among patients who may have developed disease progression while receiving, were intolerant of, or refused sorafenib. The circulating levels of cytokines, chemokines, programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and PD-L2 were correlated with response, tumor PD-L1 expression, and other clinicopathological features. RESULTS: A total of 29 patients were treated and 28 patients were evaluable for response. The most common laboratory grade 3/4 adverse events were increases in aspartate aminotransferase and/or alanine aminotransferase and serum bilirubin, which for the most part were reversible. In terms of efficacy, one patient achieved a complete response and 8 patients achieved partial responses for an overall response rate of 32%. Four other patients had stable disease. The median progression-free survival was 4.5 months and the median overall survival was 13 months. Response did not correlate with prior sorafenib therapy, PD-L1 tumor staining, or a prior history of hepatitis. Correlative studies revealed that high baseline plasma TGF-ß levels (≥200 pg/mL) significantly correlated with poor treatment outcomes after pembrolizumab. Tumor PD-L1 and plasma PD-L1/PD-1 levels were associated with plasma IFN-γ or IL-10. CONCLUSIONS: Pembrolizumab was found to demonstrate activity in patients with advanced HCC. Toxicity generally was tolerable and reversible. A set of immunological markers in blood plasma as well as PD-L1 staining indicated that baseline TGF-ß could be a predictive biomarker for response to pembrolizumab.

Optical coherence tomography imaging of melanoma skin cancer.

No consensus guidelines exist on the use of optical coherence tomography (OCT) for diagnosis of cutaneous melanoma. The objectives of this review are to provide a descriptive review of the literature on characteristics of cutaneous melanomas seen on high-definition OCT (HD-OCT), speckle variance OCT (SV-OCT), and conventional OCT and to compare their diagnostic ability with that of histopathology. A review of PubMed and Google Scholar identified all available literature on OCT in melanoma skin cancer that included all in vivo and ex vivo studies on human or human tissues and excluded all studies on non-human subjects or animal studies. Two hundred nine abstracts were considered for evaluation, 31 abstracts were selected for manuscript review, and 14 abstracts were included that met all criteria. Diagnoses of MIS and MM using HD-OCT and SV-OCT were consistently reported to correlate with histopathology. However, accuracy of diagnosis using conventional OCT varied. Most authors agreed that it was difficult to differentiate MM from benign nevi using conventional OCT. HD-OCT, SV-OCT, and conventional OCT show promise for visualizing cutaneous melanoma. The use of OCT in diagnosis of melanoma is rarely reported in the literature. There is a need to increase and standardize reporting of OCT for diagnosis of cutaneous melanoma.

Phase II trial of SOM230 (pasireotide LAR) in patients with unresectable hepatocellular carcinoma.

BACKGROUND: A phase II trial of pasireotide was performed to assess its efficacy and safety in advanced or metastatic hepatocellular carcinoma (HCC). PATIENTS AND METHODS: Patients with advanced HCC and Child-Pugh score ≤7 received pasireotide LAR 60 mg intramuscularly every 28 days. Primary endpoint was disease control rate. Secondary endpoints were time to tumor progression, response rate, treatment-related adverse events, and overall survival. Serum insulin growth factor-1 was measured before and after pasireotide. RESULTS: Twenty patients were treated and evaluable. Eighteen patients (90%) had prior therapy; 16 patients (80%) had multiple therapies. Median age was 65, 75% had Barcelona Clinic Liver Cancer stage C, and 55% had metastatic disease. The main toxicity was hyperglycemia. Rare adverse effects included reversible grade 4 elevation in alanina transaminase/aspartate transaminase in one patient. The best response was stable disease in 9 patients (45%). Median time to tumor progression for the 20 patients was 3 months, and median survival was 9 months. CONCLUSION: Pasireotide had limited clinical benefit as second-line or third-line treatment in patients with advanced or metastatic HCC. Low baseline insulin growth factor-1 level may be indicative when SOM230 treatment may be ineffective, and decreasing levels after treatment may be indicative of disease control.

Degradation of AMPK-α1 sensitizes BRAF inhibitor-resistant melanoma cells to arginine deprivation.

Melanomas harboring BRAF mutation (V600E) are known to recur frequently following treatment with BRAF inhibitors (BRAFi) despite a high initial response rate. Our previous study has uncovered that BRAFi-resistant melanoma (BR) cells are vulnerable to arginine deprivation. It has been reported that naïve melanoma cells undergo autophagy and re-express argininosuccinate synthetase 1 (ASS1) to enable them to synthesize arginine for survival when encountering arginine deprivation. Abolishing these two factors in BR cells confers sensitivity to arginine deprivation. In this report, we further demonstrated that downregulation of AMPK-α1 in BR cells is a major factor contributing to impairment of autophagy as evidenced by decreased autophagosome formation. These BR cells also showed a metabolic shift from glucose to arginine dependence, which was supported by decreased expressions of GLUT1 (glucose transporter) and hexokinase II (HKII) coupled with less glucose uptake but high levels of arginine transporter CAT-2 expression. Furthermore, silencing CAT-2 expression also distinctly attenuated BR cell proliferation. Notably, when naïve melanoma cells became BR cells by long-term exposure to BRAFi, a stepwise degradation of AMPK-α1 was initiated via ubiquitin-proteasome system (UPS). We discovered that a novel E3 ligase, RING finger 44 (RNF44), is responsible for promoting AMPK-α1 degradation in BR cells. RNF44 expression in BR cells was upregulated by transcription factor CREB triggered by hyperactivation of ERK/AKT. High levels of RNF44 corresponding to low levels of AMPK-α1 appeared in BR xenografts and melanoma tumor samples from BR and BRAFi/MEK inhibitor (MEKi)-resistant (BMR) melanoma patients. Similar to BR cells, BMR cells were also sensitive to arginine deprivation. Our study provides a novel insight into the mechanism whereby BRAFi or BRAFi/MEKi resistance drives proteasomal degradation of AMPK-α1 and consequently regulates autophagy and metabolic reprogramming in melanoma cells.

Chromatin remodeling system p300-HDAC2-Sin3A is involved in Arginine Starvation-Induced HIF-1α Degradation at the ASS1 promoter for ASS1 Derepression.

Argininosuccinate synthetase 1 (ASS1) is the key enzyme that controls biosynthesis of arginine (Arg). ASS1 is silenced in many human malignancies therefore, these tumors require extracellular Arg for growth. The Arg-degrading recombinant protein, pegylated arginine deiminase (ADI-PEG20), has been in clinical trials for targeting Arg auxotrophic tumors by Arg starvation therapy. Resistance to Arg starvation is often developed through reactivation of ASS1 expression. We previously demonstrated that ASS1 silencing is controlled by HIF-1α and Arg starvation-reactivated ASS1 is associated with HIF-1α downregulation. However, mechanisms underlying ASS1 repression and HIF-1α turnover are not known. Here, we demonstrate that interplay of p300-HDAC2-Sin3A in the chromatin remodeling system is involved in HIF-1α degradation at the ASS1 promoter. The histone acetyltransferase p300 is normally associated with the ASS1 promoter to maintain acetylated H3K14ac and H3K27ac for ASS1 silencing. Arg starvation induces p300 dissociation, allowing histone HDAC2 and cofactor Sin3A to deacetylate these histones at the ASS1 promoter, thereby facilitating HIF-1α-proteasomal complex, driven by PHD2, to degrade HIF-1α in situ. Arg starvation induces PHD2 and HDAC2 interaction which is sensitive to antioxidants. This is the first report describing epigenetic regulation of chromosomal HIF-1α turnover in gene activation that bears important implication in cancer therapy.

Autophagic Mechanism in Anti-Cancer Immunity: Its Pros and Cons for Cancer Therapy.

Autophagy, a self-eating machinery, has been reported as an adaptive response to maintain metabolic homeostasis when cancer cells encounter stress. It has been appreciated that autophagy acts as a double-edge sword to decide the fate of cancer cells upon stress factors, molecular subtypes, and microenvironmental conditions. Currently, the majority of evidence support that autophagy in cancer cells is a vital mechanism bringing on resistance to current and prospective treatments, yet whether autophagy affects the anticancer immune response remains unclear and controversial. Accumulated studies have demonstrated that triggering autophagy is able to facilitate anticancer immunity due to an increase in immunogenicity, whereas other studies suggested that autophagy is likely to disarm anticancer immunity mediated by cytotoxic T cells and nature killer (NK) cells. Hence, this contradiction needs to be elucidated. In this review, we discuss the role of autophagy in cancer cells per se and in cancer microenvironment as well as its dual regulatory roles in immune surveillance through modulating presentation of tumor antigens, development of immune cells, and expression of immune checkpoints. We further focus on emerging roles of autophagy induced by current treatments and its impact on anticancer immune response, and illustrate the pros and cons of utilizing autophagy in cancer immunotherapy based on preclinical references.

Exploiting ROS and metabolic differences to kill cisplatin resistant lung cancer.

Cisplatin resistance remains a major problem in the treatment of lung cancer. We have discovered that cisplatin resistant (CR) lung cancer cells, regardless of the signaling pathway status, share the common parameter which is an increase in reactive oxygen species (ROS) and undergo metabolic reprogramming. CR cells were no longer addicted to the glycolytic pathway, but rather relied on oxidative metabolism. They took up twice as much glutamine and were highly sensitive to glutamine deprivation. Glutamine is hydrolyzed to glutamate for glutathione synthesis, an essential factor to abrogate high ROS via xCT antiporter. Thus, blocking glutamate flux using riluzole (an amyotropic lateral sclerosis approved drug) can selectively kill CR cells in vitro and in vivo. However, we discovered here that glutathione suppression is not the primary pathway in eradicating the CR cells. Riluzole can lead to further decrease in NAD+ (nicotinamide adenine dinucleotide) and lactate dehydrogenase-A (LDHA) expressions which in turn further heightened oxidative stress in CR cells. LDHA knocked-down cells became hypersensitive to riluzole treatments and possessed increased levels of ROS. Addition of NAD+ re-stabilized LDHA and reversed riluzole induced cell death. Thus far, no drugs are available which could overcome cisplatin resistance or kill cisplatin resistant cells. CR cells possess high levels of ROS and undergo metabolic reprogramming. These metabolic adaptations can be exploited and targeted by riluzole. Riluzole may serve as a dual-targeting agent by suppression LDHA and blocking xCT antiporter. Repurposing of riluzole should be considered for future treatment of cisplatin resistant lung cancer patients.

Argininosuccinate synthetase 1 (ASS1) is a common metabolic marker of chemosensitivity for targeted arginine- and glutamine-starvation therapy.

Argininosuccinate synthetase 1 (ASS1) is the rate-limiting enzyme that catalyzes the biosynthesis of arginine (Arg). Many malignant human tumors are auxotrophic for Arg because ASS1 is silenced. ASS1 has been established as a sensor of Arg auxotrophic response and a chemosensitivity marker for Arg starvation therapy. Here, we report that ASS1 is also a sensor for glutamine (Gln)-deprivation response, and that upregulation of ASS1 expression is associated with resistance to Gln-starvation treatments. Knockdown of ASS1 expression resulted in increased sensitivity to both Arg- and Gln-starvation, whereas increased ASS1 expression by ectopic transfection is associated with resistance to both Arg- and Gln-starvation. The addition of permeable fumarate, a metabolite that bridges the tricarboxylic acid and urea cycles, resulted in downregulation of ASS1 expression and increased sensitivity to both Arg- and Gln-deprivation treatments. Mechanistically, the Gln-deprivation response, like the arginine-auxotrophic response, downregulates HIF-1α resulting in de-silencing of ASS1. Our results demonstrate that ASS1 is a common biosensor for Arg and Gln deprivation response and a shared target for Arg- and Gln-starvation therapies which have been in several current clinical trials.

Cisplatin-induced synthetic lethality to arginine-starvation therapy by transcriptional suppression of ASS1 is regulated by DEC1, HIF-1α, and c-Myc transcription network and is independent of ASS1 promoter DNA methylation.

Many human tumors require extracellular arginine (Arg) for growth because the key enzyme for de novo biosynthesis of Arg, argininosuccinate synthetase 1 (ASS1), is silenced. These tumors are sensitive to Arg-starvation therapy using pegylated arginine deiminase (ADI-PEG20) which digests extracellular Arg. Many previous studies reported that ASS1 silencing is due to epigenetic inactivation of ASS1 expression by DNA methylation, and that the demethylation agent 5-aza-deoxycytidine (Aza-dC) can induce ASS1 expression. Moreover, it was reported that cisplatin suppresses ASS1 expression through ASS1 promoter methylation, leading to synthetic lethality to ADI-PEG20 treatment. We report here that cisplatin supppresses ASS1 expression is due to upregulation of HIF-1α and downregulation of c-Myc, which function as negative and positive regulators of ASS1 expression, respectively, by reciprocal bindings to the ASS1 promoter. In contrast, we found that Aza-dC induces ASS1 expression by downregulation of HIF-1α but upregulation of c-Myc. We further demonstrated that the clock protein DEC1 is the master regulator of HIF-1α and c-Myc that regulate ASS1. cDDP upregulates DEC1, whereas Aza-dC suppresses its expression. Using two proteasomal inhibitors bortezomib and carfilzomib which induce HIF-1α accumulation, we further demonstrated that HIF-1α is involved in ASS1 silencing for the maintenance of Arg auxotrophy for targeted Arg-starvation therapy.

BRAF inhibitor resistance enhances vulnerability to arginine deprivation in melanoma.

BRAF inhibitor (BRAFi) has been used for treatment of melanomas harboring V600E mutation. Despite a high initial response rate, resistance to BRAFi is inevitable. Here, we demonstrate that BRAFi-resistant (BR) melanomas are susceptible to arginine deprivation due to inability to initiate re-expression of argininosuccinate synthetase (ASS1, a key enzyme for arginine synthesis) as well as ineffective autophagy. Autophagy and ASS1 re-expression are known to protect melanoma cells from cell death upon arginine deprivation. When melanoma cells become BR cells by long-term in vitro incubation with BRAFi, c-Myc-mediated ASS1 re-expression and the levels of autophagy-associated proteins (AMPK-α1 and Atg5) are attenuated. Furthermore, our study uncovers that downregulation of deubiquitinase USP28 which results in more active c-Myc degradation via ubiquitin-proteasome machinery is the primary mechanism for inability to re-express ASS1 upon arginine deprivation in BR cells. Overexpression of USP28 in BR cells enhances c-Myc expression and hence increases ASS1 transcription upon arginine deprivation, and consequently leads to cell survival. On the other hand, overexpression of Atg5 or AMPK-α1 in BR cells can redirect arginine deprivation-induced apoptosis toward autophagy. The xenograft models also confirm that BR tumors possess lower expression of ASS1 and are hypersensitive to arginine deprivation. These biochemical changes in BRAFi resistance which make them vulnerable to arginine deprivation can be exploited for the future treatment of BR melanoma patients.