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Supplementary Table 2. VHL Genotyping in High L-2HG Tumors.

Supplementary Figure 2. HK-2 and HEK293 cells were transiently cotransfected with the indicated siRNA and TET1 cDNA. Protein lysates were harvested and immunoblotted for the indicated proteins.

Supplementary Methods.

Supplementary Table 1. Clinical Data on Tumor Samples.

Supplementary Figure 1. A498 cells were stable transduced with the indicated construct. Protein lysates were harvested and immunoblotted for the indicated proteins.

Supplementary Figure 3. HEK293 cells were transiently transfected with either CV or L2HGDH cDNA and TET1 cDNA. Protein lysates were harvested and immunoblotted for the indicated proteins.

Purpose:Elevation of L-2-hydroxylgutarate (L-2-HG) in renal cell carcinoma (RCC) is due in part to reduced expression of L-2-HG dehydrogenase (L2HGDH). However, the contribution of L-2-HG to renal carcinogenesis and insight into the biochemistry and targets of this small molecule remains to be elucidated.Experimental Design:Genetic and pharmacologic approaches to modulate L-2-HG levels were assessed for effects on in vitro and in vivo phenotypes. Metabolomics was used to dissect the biochemical mechanisms that promote L-2-HG accumulation in RCC cells. Transcriptomic analysis was utilized to identify relevant targets of L-2-HG. Finally, bioinformatic and metabolomic analyses were used to assess the L-2-HG/L2HGDH axis as a function of patient outcome and cancer progression.Results:L2HGDH suppresses both in vitro cell migration and in vivo tumor growth and these effects are mediated by L2HGDH's catalytic activity. Biochemical studies indicate that glutamine is the predominant carbon source for L-2-HG via the activity of malate dehydrogenase 2 (MDH2). Inhibition of the glutamine-MDH2 axis suppresses in vitro phenotypes in an L-2-HG–dependent manner. Moreover, in vivo growth of RCC cells with basal elevation of L-2-HG is suppressed by glutaminase inhibition. Transcriptomic and functional analyses demonstrate that the histone demethylase KDM6A is a target of L-2-HG in RCC. Finally, increased L-2-HG levels, L2HGDH copy loss, and lower L2HGDH expression are associated with tumor progression and/or worsened prognosis in patients with RCC.Conclusions:Collectively, our studies provide biochemical and mechanistic insight into the biology of this small molecule and provide new opportunities for treating L-2-HG–driven kidney cancers.

Supplemental figures

This table reports L2HGDH mRNA expression as a function of 14q Status

Colorectal cancer (CRC) is the second leading cause of death worldwide, with 0.9 million deaths per year. The metastatic stage of the disease is identified in about 20% of cases at the first diagnosis and is associated with low patient-survival rates. Voltage-gated sodium channels (Na

Introduction: Human beings produce saliva, which is a vital secreted fluid. A significant quantity of saliva is left on the skin while biting, sucking, or licking. This saliva if effectively detected could be utilized as forensic evidence. DNA extraction and typing is complex and expensive technique; hence, ultraviolet (UV) spectroscopy could be used as an effective tool in detecting saliva. Aim: The aim was to detect the presence of dried saliva on the human skin using a UV spectrometer. Materials and Methodology: In this study, 50 volunteers deposited their saliva on their own arm. The saliva was air‑dried, then, the absorption spectra were recorded utilizing the UV spectrometer. Results: Saliva was detected with 64% of samples showing a peak at 282 nm. The technique proved to be very specific and sensitive, and it did not deteriorate the sample. Conclusion: UV‑spectroscopy is a specific and technique sensitive method that could detect the presence of saliva without deteriorating the quality of the given sample.

Introduction: Most Oral Squamous Cell Carcinoma (OSCC) is preceded by Oral Potentially Malignant Disorders (OPMDS). These disorders if diagnosed early can be prevented from converting into full blown malignancies. This points towards an ever increasing need for a more accurate, less invasive diagnostic tool to detect these lesions early. Aim: To analyse and compare the accuracy of 1% Rose Bengal (RB) dye and oral microbiopsies (using dermatologic ring curettes) with conventional scalpel biopsy in diagnosing oral epithelial dysplasias. Materials and Methods: This crosssectional pilot study, included a total number of 26 male patients, 40-60 years old, with oral white and red mucosal lesions attending the Outpatient Department (OPD) of Teerthanker Mahaveer Dental College, Moradabad, Uttar Pradesh, India. Thorough clinical examination, 1% RB staining, microbiopsy, and scalpel biopsy was performed on all included participants. Parameters assessed included clinical signs indicative of dysplasia in red and white lesions such as increase thickness, nodularity, atrophic mucosa, erosion,ulcers and change in colour of mucosa with positive history of tobacco smoking. Hyperchromatic areas owing to increase stain intake (due to increased nuclear cytoplasmic ratio) were obtained after staning with RB dye and histopathological indicators of epithelial dysplasia (cellular and architectural changes) were observed after both microbiopsy and scalpel biopsy. Chi-square test was done to compare results of 1% RB staining and microbiopsies with scalpel biopsies. Results: Total 26 male patients were included with a mean age of 50 years. There was no statistically significant difference in the accuracy of microbiopsies (p=0.913) and 1% RB dye (p=0.393) as compared to conventional scalpel biopsy in delineating the epithelial dysplastic changes associated with OPMDS. Conclusion: Oral microbiopsy, is a relatively novel, accurate, and less invasive diagnostic tool. It has proved to be as effective as scalpel biopsy in diagnosing and grading epithelial dysplasia. Through this article, it was proposed that 1% RB staining, microbiopsy, and scalpel biopsy can be used in conjugation as a part of cytology-cum-histopathology based diagnostic scheme for oral clinically suspicious lesions

Colorectal cancer (CRC) is the second leading cause of death worldwide, with 0.9 million deaths per year. The metastatic stage of the disease is identified in about 20% of cases at the first diagnosis and is associated with low patient-survival rates. Voltage-gated sodium channels (NaV) are abnormally overexpressed in several carcinomas including CRC and are strongly associated with the metastatic behavior of cancer cells. Acidification of the extracellular space by Na+/H+ exchangers (NHE) contributes to extracellular matrix degradation and cell invasiveness. In this study, we assessed the expression levels of pore-forming α-subunits of NaV channels and NHE exchangers in tumor and adjacent non-malignant tissues from colorectal cancer patients, CRC cell lines and primary tumor cells. In all cases, SCN5A (gene encoding for NaV1.5) was overexpressed and positively correlated with cancer stage and poor survival prognosis for patients. In addition, we identified an anatomical differential expression of SCN5A and SLC9A1 (gene encoding for NHE-1) being particularly relevant for tumors that originated on the sigmoid colon epithelium. The functional activity of NaV1.5 channels was characterized in CRC cell lines and the primary cells of colon tumors obtained using tumor explant methodologies. Furthermore, we assessed the performance of two new small-molecule NaV1.5 inhibitors on the reduction of sodium currents, as well as showed that silencing SCN5A and SLC9A1 substantially reduced the 2D invasive capabilities of cancer cells. Thus, our findings show that both NaV1.5 and NHE-1 represent two promising targetable membrane proteins against the metastatic progression of CRC.

Mucormycosis is an opportunistic fungal infection with a high fatality rate and is the third most common fungal infection that is invasive in nature, next to candidiasis and aspergillosis. The condition is generally vasotropic and angio-invasive in nature. It gets disseminated to a wider area locally and also exhibits a distant spread. It is usually associated with medically compromised patients. However, mucormycosis in immunocompetent individuals is gaining attention as several cases have been reported throughout the world with a high incidence of such cases being reported from the Indian subcontinent. It is attributed to the poor socio-economic status and triggered by the local trauma due to unhygienic setup or poor health care. The pathway of pathogenesis is not clearly understood in immunocompetent patients and therefore becomes a matter of great concern. Here, we report one such case of mucormycosis affecting the maxillary region following tooth extraction in a 42-year-old male.

ALS is a rare type of progressive neurological disease with unknown etiology. It results in the gradual degeneration and death of motor neurons responsible for controlling the voluntary muscles. Identification of mutations in the superoxide dismutase (SOD) 1 gene has been the most significant finding in ALS research. SOD1 abnormalities have been associated with both familial as well as sporadic ALS cases. SOD2 is a highly inducible SOD that performs in concurrence with SOD1 to detoxify ROS. Induction of SOD2 can be obtained through activation of NF-ҡBs. We previously reported that SRI-22819 increases NF-ҡB expression and activation in vitro, but it has poor ADME properties in general and has no oral bioavailability. Our initial studies were focused on direct modifications of SRI-22819. There were active compounds identified but no improvement in microsomal stability was observed. In this context, we focused on making more significant structural changes in the core of the molecule. Ataluren, an oxadiazole compound that promotes read-through and expression of dystrophin in patients with Duchenne muscular dystrophy, bears some structural similarity to SRI-22819. Thus, we synthesized a series of SRI-22819 and Ataluren (PTC124) hybrid compounds. Several compounds from this series exhibited improved activity, microsomal stability and lower calculated polar surface area (PSA). This manuscript describes the synthesis and biological evaluation of SRI-22819 analogs and its hybrid combination with Ataluren.

Purpose: Elevation of L-2-hydroxylgutarate (L-2-HG) in renal cell carcinoma (RCC) is due in part to reduced expression of L-2-HG dehydrogenase (L2HGDH). However, the contribution of L-2-HG to renal carcinogenesis and insight into the biochemistry and targets of this small molecule remains to be elucidated. Experimental Design: Genetic and pharmacologic approaches to modulate L-2-HG levels were assessed for effects on in vitro and in vivo phenotypes. Metabolomics was used to dissect the biochemical mechanisms that promote L-2-HG accumulation in RCC cells. Transcriptomic analysis was utilized to identify relevant targets of L-2-HG. Finally, bioinformatic and metabolomic analyses were used to assess the L-2-HG/L2HGDH axis as a function of patient outcome and cancer progression. Results: L2HGDH suppresses both in vitro cell migration and in vivo tumor growth and these effects are mediated by L2HGDH's catalytic activity. Biochemical studies indicate that glutamine is the predominant carbon source for L-2-HG via the activity of malate dehydrogenase 2 (MDH2). Inhibition of the glutamine-MDH2 axis suppresses in vitro phenotypes in an L-2-HG–dependent manner. Moreover, in vivo growth of RCC cells with basal elevation of L-2-HG is suppressed by glutaminase inhibition. Transcriptomic and functional analyses demonstrate that the histone demethylase KDM6A is a target of L-2-HG in RCC. Finally, increased L-2-HG levels, L2HGDH copy loss, and lower L2HGDH expression are associated with tumor progression and/or worsened prognosis in patients with RCC. Conclusions: Collectively, our studies provide biochemical and mechanistic insight into the biology of this small molecule and provide new opportunities for treating L-2-HG–driven kidney cancers.

Voltage-gated sodium channels (VGSC) are a well-established drug target for anti-epileptic, anti-arrhythmic and pain medications due to their presence and the important roles that they play in excitable cells. Recently, their presence has been recognized in non-excitable cells such as cancer cells and their overexpression has been shown to be associated with metastatic behavior in a variety of human cancers. The neonatal isoform of the VGSC subtype, Nav1.5 (nNav1.5) is overexpressed in the highly aggressive human breast cancer cell line, MDA-MB-231. The activity of nNav1.5 is known to promote the breast cancer cell invasion in vitro and metastasis in vivo, and its expression in primary mammary tumors has been associated with metastasis and patient death. Metastasis development is responsible for the high mortality of breast cancer and currently there is no treatment available to specifically prevent or inhibit breast cancer metastasis. In the present study, a 3D-QSAR model is used to assist the development of low micromolar small molecule VGSC blockers. Using this model, we have designed, synthesized and evaluated five small molecule compounds as blockers of nNav1.5-dependent inward currents in whole-cell patch-clamp experiments in MDA-MB-231 cells. The most active compound identified from these studies blocked sodium currents by 34.9 ± 6.6% at 1 μM. This compound also inhibited the invasion of MDA-MB-231 cells by 30.3 ± 4.5% at 1 μM concentration without affecting the cell viability. The potent small molecule compounds presented here have the potential to be developed as drugs for breast cancer metastasis treatment.

Background: The D-enantiomer of 2-hydroxygultarate (D-2HG), along with fumarate and succinate, are considered oncometabolites that promote tumorigenesis. Our lab has reported elevated levels of the L-enantiomer of 2-hydroxyglutare (L-2HG) in clear cell renal cell carcinoma (ccRCC), in part due to reduced expression of the enzyme L-2HG dehydrogenase (L2HGDH). Objectives: Here we evaluated the underlying biochemical mechanisms of L-2HG accumulation and characterized the contribution of the L-2HG/L2HGDH axis to tumorigenesis. Experimental strategy: We assessed the role of raised L-2HG in renal carcinogenesis via both in vitro and in vivo means. Mutagenesis and knock down approaches were applied to study involvement of loss of L2HGDH in accumulation of high L-2HG levels and associated tumor phenotypes. Metabolomics coupled with 13C tracer labeling studies were utilized to dissect the biochemical axis that promotes L-2HG accumulation in RCC cells. Results: Translational based studies demonstrate that loss of L2HGDH expression is associated with both cancer progression and worsened outcomes. In connection with our previous findings, we demonstrate that raising cellular L-2HG levels by treatment with cell permeable octyl ester of L-2HG as well as by shRNA-mediated knock down of L2HGDH in renal epithelial cells (HK-2) promotes in vitro tumor phenotypes. Concurrently, the epigenetic mark 5hmc was significantly decreased under high L-2HG levels. Further investigation with restoration of L2HGDH in RCC cells (RXF393 and A498) shows decreased L-2HG levels and suppression of in vivo tumor growth in nude mice (NU/NU). Interestingly, expression of loss-of-function mutant of L2HGDH was unable to decrease L-2HG levels and failed to suppress in vitro and in vivo tumor phenotypes. In addition, high L-2HG levels were found to upregulate epithelial-mesenchymal-transition (EMT) marker SNAIL1 and correspondingly downregulate E-cadherin. Biochemical studies demonstrate that the predominant carbon source for L-2HG in RCC is glutamine through the activity of glutaminase and malate dehydrogenase (MDH). Pharmacological inhibition of the glutamine/MDH axis by treatment of glutaminase inhibitor (CB-839) and MDH inhibitor (4k) in RCC cells reduced L-2HG levels and mitigates in vitro tumor phenotypes. Furthermore, suppression of in vitro phenotypes by CB-839 and shRNA-mediated MDH2 knockdown was rescued by concurrent treatment with octyl ester of L-2HG. Finally, restoration of L2HGDH promoted the expression of genes targeted by the polycomb repressor complex 2 (PRC2), whereas inhibition of PRC2 in high L-2HG cells suppressed tumor phenotypes. Conclusion: Collectively, our data demonstrate the biologic relevance of high L-2HG to renal carcinogenesis and reveal novel therapeutic opportunities for L-2HG driven kidney tumors. Citation Format: Sandeep Balu Shelar, Eun-hee Shim, Garrett Brinkley, Anirban Kundu, Hyeyoung Nam, Francesca Carobbio, Tyler Poston, Jubilee Tan, Daniel Benson, Dinesh Rakheja, Richard Kirkman, Yusuke Sato, Seishi Ogawa, Shilpa Dutta, Sadanandan E. Velu, David Crossman, Anja Becker, Conrad Kunick, Sunil Sudarshan. L-2HG/ L2HGDH axis as therapeutic target for kidney cancer [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 5482.

The ocean continues to provide a plethora of unique scaffolds capable of remarkable biological applications. A large number of pyrroloiminoquinone alkaloids, including discorhabdins, epinardins, batzellines, makaluvamines, and veiutamine, have been isolated from various marine organisms. A class of pyrroloiminoquinone-related alkaloids, known as bispyrroloquinones, is the focus of this review article. This family of marine alkaloids, which contain an aryl substituted bispyrroloquinone ring system, includes three subclasses of alkaloids namely, wakayin, tsitsikammamines A-B, and zyzzyanones A-D. Both wakayin and the tsitsikammamines contain a tetracyclic fused bispyrroloiminoquinone ring system, while zyzzyanones contain a fused tricyclic bispyrroloquinone ring system. The unique chemical structures of these marine natural products and their diverse biological properties, including antifungal and antimicrobial activity, as well as the potent, albeit generally nonspecific and universal cytotoxicities, have attracted great interest of synthetic chemists over the past three decades. Tsitsikammamines, wakayin, and several of their analogs show inhibition of topoisomerases. One additional possible mechanism of anticancer activity of tsitsikammamines analogs that has been discovered recently is through the inhibition of indoleamine 2, 3-dioxygenase, an enzyme involved in tumoral immune resistance. This review discusses the isolation, synthesis, and evaluation of bioactivities of bispyrroloquinone alkaloids and their analogs.

Through unbiased metabolomics, we identified elevations of the metabolite 2-hydroxyglutarate (2HG) in renal cell carcinoma (RCC). 2HG can inhibit 2-oxoglutaratre (2-OG)–dependent dioxygenases that mediate epigenetic events, including DNA and histone demethylation. 2HG accumulation, specifically the d enantiomer, can result from gain-of-function mutations of isocitrate dehydrogenase (IDH1, IDH2) found in several different tumors. In contrast, kidney tumors demonstrate elevations of the l enantiomer of 2HG (l-2HG). High-2HG tumors demonstrate reduced DNA levels of 5-hydroxymethylcytosine (5hmC), consistent with 2HG-mediated inhibition of ten-eleven translocation (TET) enzymes, which convert 5-methylcytosine (5mC) to 5hmC. l-2HG elevation is mediated in part by reduced expression of l-2HG dehydrogenase (L2HGDH). L2HGDH reconstitution in RCC cells lowers l-2HG and promotes 5hmC accumulation. In addition, L2HGDH expression in RCC cells reduces histone methylation and suppresses in vitro tumor phenotypes. Our report identifies l-2HG as an epigenetic modifier and putative oncometabolite in kidney cancer. Significance: Here, we report elevations of the putative oncometabolite l-2HG in the most common subtype of kidney cancer and describe a novel mechanism for the regulation of DNA 5hmC levels. Our findings provide new insight into the metabolic basis for the epigenetic landscape of renal cancer. Cancer Discov; 4(11); 1290–8. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 1243

Pyrroloquinone ring systems are important structural units present in many biologically active molecules including a number of marine alkaloids. For example, they are found in a series of marine metabolites, such as tsitsikammamines, zyzzyanones, wakayin, and terreusinone. Several of these alkaloids have exhibited antimicrobial, antimalarial, antifungal, antitumor, and photoprotecting activities. Synthesis of pyrroloquinone unit is the key step in the synthesis of many of these important organic molecules. Here, we present a ceric (IV) ammonium nitrate (CAN) mediated oxidative free radical cyclization reaction of 1,3-dicarbonyl compounds with aminoquinones as a facile methodology for making various substituted pyrroloquinones. 1,3-dicarbonyl compounds used in this study are ethyl acetoacetate, acetylacetone, benzoyl acetone, andN,N-dimethyl acetoacetamide. The aminoquinones used in this study are 2-(benzylamino)naphthalene-1,4-dione and 6-(benzylamino)-1-tosyl-1H-indole-4,7-dione. The yields of the synthesized pyrroloquinones ranged from 23–91%.

The marine environment is host to unparalleled biological and chemical diversity, making it an attractive resource for the discovery of new therapeutics for a plethora of diseases. Compounds that are extracted from cyanobacteria are of special interest due to their unique structural scaffolds and capacity to produce potent pharmaceutical and biotechnological traits. Calothrixins A and B are two cyanobacterial metabolites with a structural assembly of quinoline, quinone, and indole pharmacophores. This review surveys recent advances in the synthesis and evaluation of the biological activities of calothrixins. Due to the low isolation yields from the marine source and the promise this scaffold holds for anticancer and antimicrobial drugs, organic and medicinal chemists around the world have embarked on developing efficient synthetic routes to produce calothrixins. Since the first review appeared in 2009, 11 novel syntheses of calothrixins have been published in the efforts to develop methods that contain fewer steps and higher-yielding reactions. Calothrixins have shown their potential as topoisomerase I poisons for their cytotoxicity in cancer. They have also been observed to target various aspects of RNA synthesis in bacteria. Further investigation into the exact mechanism for their bioactivity is still required for many of its analogs.

Kinase inhibitors are among the fastest growing class of anti-cancer therapies. One family of kinases that has recently gained attention as a target for treating malignant disorders is the Tec kinase family. Evidence has been published that one member of this family; the Bmx kinase, may play a role in the pathogenesis of glioblastoma, prostate, breast and lung cancer. Bmx has also shown potential as an anti-vascular therapy in combination with radiation or as a sensitizer to chemotherapeutic agents. Therefore, several companies such as Pharmacyclics, Avila Therapeutics, Merck and Co., Metaproteomics, IRM, and Moerae Matrix have developed compounds or peptides that function as Bmx kinase inhibitors. These companies have subsequently been issued patents for these inhibitors. Additionally, it has been shown that current clinical stage EGFR inhibitors can irreversibly inhibit Bmx, suggesting these compounds might be rapidly moved to clinical trials for other malignancies. This review will discuss current patents issued since 2009 that contain data specifically on inhibition of the Bmx kinase, and will also discuss the scientific literature that suggests their potential application as therapeutics in the treatment of the aforementioned malignancies.