Preparation and evaluation of 99mTc(V)-DMSA complex: studies in medullary carcinoma of thyroid

Consequent to the promising results reported with 99mTc(V)-DMSA for imaging certain types of soft tissue tumors, we have developed methods to prepare this radiopharmaceutical in three ways: from freshly prepared reagents, through the use of a two component kit and use of the standard renal DMSA kit by a modified recipe. The 99mTc(V)-DMSA complex has been subjected to paper electrophoretic and chromatographic procedures and also biodistribution studies. The distinctly different behaviour of this new product compared to that of the well known renal DMSA complex has been clearly established. Scintiimaging in a preliminary clinical trial in patients with medullary carcinoma of the thyroid has been encouraging.     

Horizontal transfer of domains in ssrA gene among Enterobacteriaceae

The tmRNA (transfer messenger RNA), encoded by ssrA gene, is involved in rescuing of stalled ribosomes by a process called trans-translation. Additionally, regions of the ssrA gene (coding for tmRNA) were reported to serve as integration sites for various bacteriophages. Though variations in ssrA genes were reported, their functional relevance is less studied. In this study, we investigated the horizontal gene transfer (HGT) of ssrA among the members of Enterobacteriaceae. This was done by predicting recombination signals in ssrA gene (belonging to Enterobacteriaceae) using RDP5 (Recombination Detection Program 5). Our results revealed 7 recombination signals in ssrA gene belonging to different species. We further showed that the recombination signals were more in the domains present in the 3′ end than 5′ end of tmRNA. Of note, the mRNA region was reported in many recombination signals. Further, members belonging to genera Yersinia, Erwinia, Dickeya and Enterobacter were highly represented in the recombination signals. Sequence analysis revealed the presence of integration sites for different class of bacteriophages in ssrA gene. The locations of phage recognition sites are comparable with recombination signals. Taken together, our results revealed a diverse nature of HGT and recombination which possibly due to transduction mediated by phages.     

Common and rare alleles of the serotonin transporter gene,SLC6A4, associated with Tourette's disorder

To evaluate the hypothesis that functionally over‐expressing alleles of the serotonin transporter (SERT) gene (solute carrier family 6, member 4, SLC6A4) are present in Tourette's disorder (TD), just as we previously observed in obsessive compulsive disorder (OCD), we evaluated TD probands (N = 151) and controls (N = 858). We genotyped the refined SERT‐linked polymorphic region 5‐HTTLPR/rs25531 and the associated rs25532 variant in the SLC6A4 promoter plus the rare coding variant SERT isoleucine‐to‐valine at position 425 (I425V). The higher expressing 5‐HTTLPR/rs25531 L_A allele was more prevalent in TD probands than in controls (χ² = 5.75; P = 0.017; odds ratio [OR], 1.35); and, in a secondary analysis, surprisingly, it was significantly more frequent in probands who had TD alone than in those who had TD plus OCD (Fisher's exact test; P = 0.0006; OR, 2.29). Likewise, the higher expressing L_AC haplotype (5‐HTTLPR/rs25531/rs25532) was more frequent in TD probands than in controls (P = 0.024; OR, 1.33) and also in the TD alone group versus the TD plus OCD group (P = 0.0013; OR, 2.14). Furthermore, the rare gain‐of‐function SERT I425V variant was observed in 3 male siblings with TD and/or OCD and in their father. Thus, the cumulative count of SERT I425V becomes 1.57% in OCD/TD spectrum conditions versus 0.15% in controls, with a recalculated, family‐adjusted significance of χ² = 15.03 (P < 0.0001; OR, 9.0; total worldwide genotyped, 2914). This report provides a unique combination of common and rare variants in one gene in TD, all of which are associated with SERT gain of function. Thus, altered SERT activity represents a potential contributor to serotonergic abnormalities in TD. The present results call for replication in a similarly intensively evaluated sample. © 2013 Movement Disorder Society     

Infantile Fibrosarcoma With NTRK3–ETV6 Fusion Successfully Treated With the Tropomyosin‐Related Kinase Inhibitor LOXO‐101

Infantile fibrosarcoma (IFS) is a rare pediatric cancer typically presenting in the first 2 years of life. Surgical resection is usually curative and chemotherapy is active against gross residual disease. However, when recurrences occur, therapeutic options are limited. We report a case of refractory IFS with constitutive activation of the tropomyosin‐related kinase (TRK) signaling pathway from an ETS variant gene 6–neurotrophin 3 receptor gene (ETV6–NTRK3) gene fusion. The patient enrolled in a pediatric Phase 1 trial of LOXO‐101, an experimental, highly selective inhibitor of TRK. The patient experienced a rapid, radiographic response, demonstrating the potential for LOXO‐101 to provide benefit for IFS harboring NTRK gene fusions.     

Catastrophic ATP loss underlies a metabolic combination therapy tailored for MYCN-amplified neuroblastoma

MYCN-amplified neuroblastoma is a lethal subset of pediatric cancer. MYCN drives numerous effects in the cell, including metabolic changes that are critical for oncogenesis. The understanding that both compensatory pathways and intrinsic redundancy in cell systems exists implies that the use of combination therapies for effective and durable responses is necessary. Additionally, the most effective targeted therapies exploit an “Achilles’ heel” and are tailored to the genetics of the cancer under study. We performed an unbiased screen on select metabolic targeted therapy combinations and correlated sensitivity with over 20 subsets of cancer. We found that MYCN-amplified neuroblastoma is hypersensitive to the combination of an inhibitor of the lactate transporter MCT1, AZD3965, and complex I of the mitochondrion, phenformin. Our data demonstrate that MCT4 is highly correlated with resistance to the combination in the screen and lowly expressed in MYCN-amplified neuroblastoma. Low MCT4 combines with high expression of the MCT2 and MCT1 chaperone CD147 in MYCN-amplified neuroblastoma, altogether conferring sensitivity to the AZD3965 and phenformin combination. The result is simultaneous disruption of glycolysis and oxidative phosphorylation, resulting in dramatic disruption of adenosine triphosphate (ATP) production, endoplasmic reticulum stress, and cell death. In mouse models of MYCN-amplified neuroblastoma, the combination was tolerable at concentrations where it shrank tumors and did not increase white-blood-cell toxicity compared to single drugs. Therefore, we demonstrate that a metabolic combination screen can identify vulnerabilities in subsets of cancer and put forth a metabolic combination therapy tailored for MYCN-amplified neuroblastoma that demonstrates efficacy and tolerability in vivo.

Despite their relative rarity compared to blood cancers, solid-tumor pediatric cancers are now the leading cause of pediatric cancer-related deaths. Among the most deadly is high-risk neuroblastoma (NB): amplification of MYCN confers high risk and is the clear driver of NB in these cancers (1). As such, MYCN remains the most important drug target in NB and one of the most important in pediatric cancer. Unfortunately, direct chemical targeting of MYCN has not yet been successful, and despite advancements in anti-GD2 immunotherapy (2), alternate ways of targeting MYCN-amplified NB may be needed to successfully treat this cancer.One approach is to find tumor-specific vulnerabilities, which are exploitable pharmacologically. Many efforts, including ours (3), have exhaustively looked for kinase inhibitors with particular efficacy in MYCN-amplified NBs. However, the emerging picture is a lack of kinase inhibitor efficacy in MYCN-amplified NB. Other vulnerabilities may be classified under the broad category of drugs targeting epigenetic modifiers. For example, using a CRISPR/Cas9 screen, Stegmaier and colleagues demonstrated that MYCN-amplified NB may be susceptible to targeting the H3K27me methylase EZH2 (4); in a different study, they demonstrated the susceptibility of MYCN-amplified NB to the combination of BRD4 inhibitors with CDK7 inhibitors (5). In addition, Thiele and colleagues (6) demonstrated high-risk NBs were susceptible to inhibition of the lysine methyltransferase SETD8. As promising as these data are, it remains unknown whether tolerability and/or clinical activity in MYCN-amplified NB will occur and SETD8, BRD4, and CDK7 inhibitors so far are not in the pediatric clinic. Cell death inducers constitute a third category. To this point, we recently uncovered a susceptibility of MYCN-amplified NB to the BCL-2 inhibitor venetoclax (3), confirmed by others (7). There, MYCN-driven NOXA expression sensitizes cells to venetoclax (3). Venetoclax is now in early phase trials in pediatric patients including those with NB ({"type":"clinical-trial","attrs":{"text":"NCT03236857","term_id":"NCT03236857"}}NCT03236857). It remains to be seen whether or not it will elicit responses in NB patients as a single agent.A fourth distinct category of therapeutic strategies to indirectly target oncogenes is through metabolism targeting, involving the growing coterie of drugs targeting the pathways fulfilling the high-energy demands of cancer cells. A major energy currency in cells is adenosine triphosphate (ATP). The Warburg effect describes the propensity of cancer cells (and highly proliferating normal cells) to produce ATP in the presence of oxygen with the less efficient, extramitochondrial glycolysis, as opposed to the more efficient mitochondria-based oxidative phosphorylation occurring in most noncancerous cells (8). The mechanistic explanation of the Warburg effect and how it might benefit cancer cells has been revised dramatically over the years. It was originally proposed that mitochondria from cancer cells were defective and lacked oxidative phosphorylation capabilities (9); on the contrary, emerging data show that many cancers rely on oxidative phosphorylation to facilitate the generation of ATP (8, 10). Interestingly, while amplified MYCN directly regulates the expression of many of the key glycolytic enzymes and as such contributes to the Warburg effect (11, 12), a study utilizing a Seahorse respirator demonstrated that a MYCN-amplified NB cell line favored oxidative phosphorylation over glycolysis for the metabolic needs, while the reverse was true for a MYCN wild-type NB cell line (13). In an independent study, MYCN was associated with higher glycolytic flux and oxidative phosphorylation and conferred sensitivity to fatty acid oxidation disruption (12). Overall, since c-MYC, which shares ∼40% binding homology to DNA-binding sites throughout the genome with MYCN, has been extensively characterized as a metabolic master regulator (14, 15), it is likely there are other MYCN-driven metabolic processes that may represent significant drug targets.Monocarboxylate transporters (MCTs) consist of four members (MCT1–4) in mammalian cells. Among their most critical substrates are lactate and pyruvate; MCT1 and MCT4 are responsible for lactate export across the plasma membrane to the extracellular space (16). AZD3965 (17) (AstraZeneca) is the first in-class–specific MCT1/2 dual inhibitor and is currently in early phase trials for diverse cancers; however, other inhibitors from different companies have recently been developed as well (18). Of note, AZD3965 has demonstrated good tolerability in diverse patients (clinical trial number {"type":"clinical-trial","attrs":{"text":"NCT01791595","term_id":"NCT01791595"}}NCT01791595). Although rare (65 cases/100,000 person-years), lactic acidosis led to the market retrieval of phenformin in America (19), yet phenformin remains in use as a type II antidiabetic drug in Europe, functioning centrally as a mitochondrial complex I electron transport chain (ETC) inhibitor. Phenformin reduces both glycolytic intermediates and pyruvate, increases shunting of glucose-derived carbon (increasing total lactate production), and markedly reduces tricarboxylic acid cycle intermediates (20). Indeed, there has been a recent resurgence in interest in the use of phenformin to treat cancer. For example, in BRAF mutant melanoma, phenformin sensitized cells to BRAF inhibitor through cooperative suppression of the metabolic sensor pathway mTORC1 (21). These preclinical data have led to a clinical trial of phenformin in combination with BRAF inhibitor in BRAF mutant melanoma ({"type":"clinical-trial","attrs":{"text":"NCT03026517","term_id":"NCT03026517"}}NCT03026517). Overall, while targeting individual metabolic pathways has demonstrated some preclinical success in different cancer models, it is limited with significant redundancy in pathways to generate ATP and regenerate NAD+ (22). We therefore assessed potential combination therapies involving metabolic targeting drugs to identify a strategy for MYCN-amplified NB.