Effect of sulfasalazine on human neuroblastoma: Analysis of sepiapterin reductase (SPR) as a new therapeutic target

Background: Neuroblastoma (NB) is an aggressive childhood malignancy in children up to 5 years of age. High-stage tumors frequently relapse even after aggressive multimodal treatment, and then show therapy resistance, typically resulting in patient death. New molecular-targeted compounds that effectively suppress tumor growth and prevent relapse with more efficacy are urgently needed. We and others previously showed that polyamines (PA) like spermidine and spermine are essential for NB tumorigenesis and that DFMO, an inhibitor of the key PA synthesis gene product ODC, is effective both in vitro and in vivo, securing its evaluation in NB clinical trials. To find additional compounds interfering with PA biosynthesis, we tested sulfasalazine (SSZ), an FDA-approved salicylate-based anti-inflammatory and immune-modulatory drug, recently identified to inhibit sepiapterin reductase (SPR). We earlier presented evidence for a physical interaction between ODC and SPR and we showed that RNAi-mediated knockdown of SPR expression significantly reduced native ODC enzyme activity and impeded NB cell proliferation. Methods: Human NB mRNA expression datasets in the public domain were analyzed using the R2 platform. Cell viability, isobologram, and combination index analyses as a result of SSZ treatment with our without DFMO were carried out in NB cell cultures. Molecular protein-ligand docking was achieved using the GRAMM algorithm. Statistical analyses were performed with the Kruskal-Wallis test, 2log Pearson test, and Student's t test. Results: In this study, we show the clinical relevance of SPR in human NB tumors. We found that high SPR expression is significantly correlated to unfavorable NB characteristics like high age at diagnosis, MYCN amplification, and high INSS stage. SSZ inhibits the growth of NB cells in vitro, presumably due to the inhibition of SPR as predicted by computational docking of SSZ into SPR. Importantly, the combination of SSZ with DFMO produces synergistic antiproliferative effects in vitro. Conclusions: The results suggest the use of SSZ in combination with DFMO for further experiments, and possible prioritization as a novel therapy for the treatment of NB patients

Metabolism within the tumor microenvironment and its implication on cancer progression: an ongoing therapeutic target

Since reprogramming energy metabolism is considered a new hallmark of cancer, tumor metabolism is again in the spotlight of cancer research. Many studies have been carried out and many possible therapies have been developed in the last years. However, tumor cells are not alone. A series of extracellular components and stromal cells, such as endothelial cells, cancer-associated fibroblasts, tumor-associated macrophages and tumor-infiltrating T cells, surround tumor cells in the so-called tumor microenvironment. Metabolic features of these cells are being studied in deep in order to find relationships between metabolism within the tumor microenvironment and tumor progression. Moreover, it cannot be forgotten that tumor growth is able to modulate host metabolism and homeostasis, so that tumor microenvironment is not the whole story. Importantly, the metabolic switch in cancer is just a consequence of the flexibility and adaptability of metabolism and should not be surprising. Treatments of cancer patients with combined therapies including anti-tumor agents with those targeting stromal cell metabolism, anti-angiogenic drugs and/or immunotherapy are being developed as promising therapeutics.Mª Carmen Ocaña is recipient of a predoctoral FPU grant from the Spanish Ministry of Education, Culture and Sport. Supported by grants BIO2014-56092-R (MINECO and FEDER), P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript

Immunometabolism: A ‘Hot’ Switch for ‘Cold’ Pediatric Solid Tumors

Despite the success of immunotherapies in adult solid cancers and pediatric hematological malignancies, limited progress has been made towards implementing these strategies in pediatric solid tumors. These tumors exhibit a high potential to escape antitumor immunity, making them difficult to target by current immunotherapies. This review highlights the altered metabolic pathways in pediatric solid tumors that promote immune escape, and discusses current novel strategies targeting these pathways. We further explore how these strategies could be applied to potentiate immunotherapies for pediatric solid cancers and pose key questions yet to be addressed. Translational challenges to facilitate clinical application of antimetabolic strategies through personalized medicine are identified. We propose preclinical testing of antimetabolic approaches in combination with immunotherapies for pediatric solid cancers

백금 민감성 및 저항성 난소암에 대한 새로운 치료 전략으로서 DFMO를 이용한 폴리아민 경로의 억제

학위논문(박사) -- 서울대학교대학원 : 의과대학 의학과, 2023. 8. 김용범.There is a persistent need for more effective anti-ovarian-cancer drugs with lower toxicity. One anticancer strategy involves the comprehension of molecular functions and the targeting of polyamine metabolism. DFMO, an irreversible ODC inhibitor, has demonstrated anticancer activity by suppressing polyamine synthesis while maintaining a high safety profile. Previously, we discovered that DFMO induced apoptosis in an ovarian cancer cell line, SKOV-3, by increasing AP-1 and JNK phosphorylation expression, either alone or in combination with cisplatin. As a continuation of the previous study, this investigation aims to ascertain the effect of DFMO alone or in combination with conventional chemotherapy, cisplatin, on other ovarian cancer cell lines, including those resistant to cisplatin. In addition, inhibition of tumor growth by DFMO with or without cisplatin will be evaluated in a mouse model. As a result, ovarian cancer cells other than SKOV-3 were found to have a comparable effect in this study. In a mouse xenograft model, DFMO alone or combined with cisplatin suppressed tumorigenesis and altered the mRNA expression of polyamine signaling, angiogenesis, cancer stemness, and apoptosis markers. DFMO also caused apoptosis in cisplatin-resistant ovarian cancer cells by inhibiting polyamine synthesis signaling. These findings indicate that DFMO treatment, either alone or in combination with cisplatin, could be a promising treatment for ovarian cancer.더 낮은 독성을 가진 더 효과적인 난소암 치료 약물에 대한 지속적인 요구가 있다. 한 가지 항암 전략은 분자 기능의 이해와 폴리아민 대사의 표적화를 포함한다. 비가역적인 ODC 억제제인 DFMO는 높은 안전성 프로필을 유지하면서 폴리아민 합성을 억제함으로써 항암 활성을 입증했다. 이전에 우리는 DFMO가 단독으로 또는 시스플라틴과 함께 AP-1 및 JNK 인산화 발현을 증가시킴으로써 난소암 세포주인 SKOV-3에서 세포사멸을 유도한다는 것을 발견했다. 이전 연구의 연속으로, 이 연구는 시스플라틴에 내성이 있는 세포를 포함하여 다른 난소암 세포주에 대한 DFMO 단독 또는 기존 화학 요법인 시스플라틴과의 병합 효과를 확인하는 것을 목표로 하고자 한다. 또한, DFMO 단독 또는 시스플라틴과의 병합에 의한 종양 성장 억제를 마우스 모델에서 평가하였다. 본 연구 결과, SKOV-3 이외의 난소암 세포에서도 비슷한 효과를 나타내는 것으로 나타났다. 마우스 이종이식 모델에서도 DFMO 단독 또는 시스플라틴과의 병합이 종양 형성을 억제하고 폴리아민 신호, 혈관신생, 암 줄기세포 및 세포자살 마커의 mRNA 발현을 바꾸는 것을 확인했다. 또한 DFMO는 폴리아민 합성 신호를 억제하여 시스플라틴 내성 난소암 세포에서 세포사멸을 일으켰다. 이러한 결과는 DFMO 치료 단독 또는 시스플라틴과의 조합이 난소암에 대한 유망한 치료가 될 수 있음을 시사한다.1. Introduction 1 2. Materials and Methods 4 3. Results 11 4. Discussion 26 Bibliography 30 Abstract in Korean 34박

Oligoamine analogues in combination with 2-difluoromethylornithine synergistically induce re-expression of aberrantly silenced tumour-suppressor genes

Epigenetic gene silencing is an important mechanism in the initiation and progression of cancer. Abnormal DNA CpG island hypermethylation and histone modifications are involved in aberrant silencing of tumour-suppressor genes. LSD1 (lysine-specific demethylase 1) was the first enzyme identified to specifically demethylate H3K4 (Lys4 of histone H3). Methylated H3K4 is an important mark associated with transcriptional activation. The flavin adenine dinucleotide-binding amine oxidase domain of LSD1 is homologous with two polyamine oxidases, SMO (spermine oxidase) and APAO (N1-acetylpolyamine oxidase). We have demonstrated previously that long-chain polyamine analogues, the oligoamines, are inhibitors of LSD1. In the present paper we report the synergistic effects of specific oligoamines in combination with DFMO (2-difluoromethylornithine), an inhibitor of ornithine decarboxylase, in human colorectal cancer cells. DFMO treatment depletes natural polyamines and increases the uptake of exogenous polyamines. The combination of oligoamines and DFMO results in a synergistic re-expression of aberrantly silenced tumour-suppressor genes, including SFRP2 (secreted frizzled-related protein 2), which encodes a Wnt signalling pathway antagonist and plays an anti-tumorigenic role in colorectal cancer. The treatment-induced re-expression of SFRP2 is associated with increased H3K4me2 (di-methyl H3K4) in the gene promoter. The combination of LSD1-inhibiting oligoamines and DFMO represents a novel approach to epigenetic therapy of cancer

A G316A polymorphism in the ornithine decarboxylase gene promoter modulates MYCN-driven childhood neuroblastoma

Simple Summary Neuroblastoma is a devasting childhood cancer in which multiple copies (amplification) of the cancer-causing gene MYCN strongly predict poor outcome. Neuroblastomas are reliant on high levels of cellular components called polyamines for their growth and malignant behavior, and the gene regulating polyamine synthesis is called ODC1. ODC1 is often coamplified with MYCN, and in fact is regulated by MYCN, and like MYCN is prognostic of poor outcome. Here we studied a naturally occurring genetic variant or polymorphism that occurs in the ODC1 gene, and used gene editing to demonstrate the functional importance of this variant in terms of ODC1 levels and growth of neuroblastoma cells. We showed that this variant impacts the ability of MYCN to regulate ODC1, and that it also influences outcome in neuroblastoma, with the rarer variant associated with a better survival. This study addresses the important topic of genetic polymorphisms in cancer. Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein

A g316a polymorphism in the ornithine decarboxylase gene promoter modulates mycn‐driven childhood neuroblastoma

Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR‐cas9 technology was used to investigate the effects of AG clones from wildtype MYCN‐amplified SK‐N‐BE(2)‐C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neu-roblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN‐amplified and non‐amplified patients, re-spectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele‐specific regulation by the MYCN oncoprotein

Arginine enzymatic deprivation and diet restriction for cancer treatment

Recent findings in amino acid metabolism and the differences between normal, healthy cells and neoplastic cells have revealed that targeting single amino acid metabolic enzymes in cancer therapy is a promising strategy for the development of novel therapeutic agents. Arginine is derived from dietary protein intake, body protein breakdown, or endogenous de novo arginine production and several studies have revealed disturbances in its synthesis and metabolism which could enhance or inhibit tumor cell growth. Consequently, there has been an increased interest in the arginine-depleting enzymes and dietary deprivation of arginine and its precursors as a potential antineoplastic therapy. This review outlines the most recent advances in targeting arginine metabolic pathways in cancer therapy and the different chemo- and radio-therapeutic approaches to be co-applied

Polyamine Blocking Therapy as a Novel Therapeutic Strategy for High-risk Paediatric Cancers

Leukaemia is the most common cancer in children, whereas neuroblastoma is the most common extracranial solid tumour of childhood. Outcomes for patients diagnosed with high-risk subtypes of these cancers remain suboptimal, necessitating the development of better therapies. Polyamines are essential for many cellular functions and are frequently upregulated in cancers, thus starving cancer cells by targeting their polyamine dependency through blocking polyamine synthesis has been under investigation as a strategy for cancer treatment. As it was discovered that solid cancer cells could counteract the effects of polyamine synthesis inhibition by increasing polyamine uptake from the extracellular environment, it became clear that simultaneous targeting of both polyamine biosynthesis and uptake would be instrumental for polyamine depletion to become an effective treatment for solid cancers. As a result, the combination of DFMO, a polyamine synthesis inhibitor, and AMXT 1501, a polyamine transport inhibitor, is currently being tested in early phase trials for adult solid tumours (NCT03536728) and in patients with Diffuse or High Grade Glioma (NCT05717153). However, very little is known about the mechanisms that govern polyamine uptake by cancer cells and while polyamine depletion is under investigation as a treatment for solid cancers, its potential for the treatment of acute leukaemias is unknown. This study identified ATP13A3 as a key polyamine transporter in neuroblastoma by the demonstration that silencing of ATP13A3 significantly reduced basal polyamine uptake and prevented DFMO-induced polyamine uptake compensation. ATP13A3 silencing suppressed neuroblastoma cell growth and potentiated the effects of DFMO in suppressing neuroblastoma cell colony formation, providing evidence that ATP13A3 holds potential as a novel candidate target for neuroblastoma treatment. In addition, this study discovered that similar to solid cancer cells, acute leukaemia cells are also addicted to polyamines and dependent on both polyamine synthesis and uptake. The combination of DFMO and AMXT 1501 synergistically reduced acute leukaemia cell growth and delayed progression in two animal models of high-risk infant KMT2A-rearranged leukaemia. This study thereby provides evidence that dual targeting of polyamine synthesis and uptake can be further exploited in preclinical and clinical investigations in acute leukaemia to develop better therapeutics for this detrimental disease