Targeting prostate tumor low-molecular weight tyrosine phosphatase for oxidation-sensitizing therapy.

Protein tyrosine phosphatases (PTPs) play major roles in cancer and are emerging as therapeutic targets. Recent reports suggest low-molecular weight PTP (LMPTP)-encoded by the ACP1 gene-is overexpressed in prostate tumors. We found ACP1 up-regulated in human prostate tumors and ACP1 expression inversely correlated with overall survival. Using CRISPR-Cas9-generated LMPTP knockout C4-2B and MyC-CaP cells, we identified LMPTP as a critical promoter of prostate cancer (PCa) growth and bone metastasis. Through metabolomics, we found that LMPTP promotes PCa cell glutathione synthesis by dephosphorylating glutathione synthetase on inhibitory Tyr270. PCa cells lacking LMPTP showed reduced glutathione, enhanced activation of eukaryotic initiation factor 2-mediated stress response, and enhanced reactive oxygen species after exposure to taxane drugs. LMPTP inhibition slowed primary and bone metastatic prostate tumor growth in mice. These findings reveal a role for LMPTP as a critical promoter of PCa growth and metastasis and validate LMPTP inhibition as a therapeutic strategy for treating PCa through sensitization to oxidative stress.

Novel two-tiered screening approach identifies synergistic combinations of natural compounds for prostate cancer prevention and treatment.

Prostate cancer (PCa) is the second most common cancer type among American men and it is estimated that in 2023, 34,700 men will die from PCa. Since it can take a considerable amount of time for the disease to progress to clinically evident cancer, there is ample opportunity for effective chemopreventive strategies to be applied for the successful management of PCa progression. In the current study, we have developed a two-tiered metabolomics-based screen to identify synergistic combinations of phytochemicals for PCa chemoprevention. This involves an initial screen for ATP depletion in PCa cells followed by a targeted screen for blocking glutamine uptake in the same cells. One of the phytochemical combinations (enoxolone [ENO] + silibinin [SIL]), identified via this screen, was examined for effects on PCa cell survival, oncogenic signaling and tumor growth in vivo. This combination was found to synergistically reduce cell survival, colony formation and cell cycle progression of PCa cell lines to a greater extent than either agent alone. The combination of ENO and SIL also synergistically reduced tumor growth when administered ad libitum through the diet in a HMVP2 allograft PCa tumor model. Treatment with the combination also significantly reduced STAT3 and mTORC1 signaling pathways in mouse and human PCa cells while significantly reducing levels of critical cell cycle regulatory proteins, contributing to the synergistic inhibition of tumor growth observed. Collectively, the current results demonstrate a novel approach to identifying synergistic combinations of phytochemicals for chemoprevention of PCa and possibly other cancers.

Generation of a Yeast Cell Model Potentially Useful to Identify the Mammalian Mitochondrial N-Acetylglutamate Transporter.

The human mitochondrial carrier family (MCF) consists of 53 members. Approximately one-fifth of them are still orphans of a function. Most mitochondrial transporters have been functionally characterized by reconstituting the bacterially expressed protein into liposomes and transport assays with radiolabeled compounds. The efficacy of this experimental approach is constrained to the commercial availability of the radiolabeled substrate to be used in the transport assays. A striking example is that of N-acetylglutamate (NAG), an essential regulator of the carbamoyl synthetase I activity and the entire urea cycle. Mammals cannot modulate mitochondrial NAG synthesis but can regulate the levels of NAG in the matrix by exporting it to the cytosol, where it is degraded. The mitochondrial NAG transporter is still unknown. Here, we report the generation of a yeast cell model suitable for identifying the putative mammalian mitochondrial NAG transporter. In yeast, the arginine biosynthesis starts in the mitochondria from NAG which is converted to ornithine that, once transported into cytosol, is metabolized to arginine. The deletion of ARG8 makes yeast cells unable to grow in the absence of arginine since they cannot synthetize ornithine but can still produce NAG. To make yeast cells dependent on a mitochondrial NAG exporter, we moved most of the yeast mitochondrial biosynthetic pathway to the cytosol by expressing four E. coli enzymes, argB-E, able to convert cytosolic NAG to ornithine. Although argB-E rescued the arginine auxotrophy of arg8∆ strain very poorly, the expression of the bacterial NAG synthase (argA), which would mimic the function of a putative NAG transporter increasing the cytosolic levels of NAG, fully rescued the growth defect of arg8∆ strain in the absence of arginine, demonstrating the potential suitability of the model generated.

Glutaminase inhibition in combination with azacytidine in myelodysplastic syndromes: Clinical efficacy and correlative analyses.

Malignancies can become reliant on glutamine as an alternative energy source and as a facilitator of aberrant DNA methylation, thus implicating glutaminase (GLS) as a potential therapeutic target. We demonstrate preclinical synergy of telaglenastat (CB-839), a selective GLS inhibitor, when combined with azacytidine (AZA), in vitro and in vivo, followed by a phase Ib/II study of the combination in patients with advanced MDS. Treatment with telaglenastat/AZA led to an ORR of 70% with CR/mCRs in 53% patients and a median overall survival of 11.6 months. scRNAseq and flow cytometry demonstrated a myeloid differentiation program at the stem cell level in clinical responders. Expression of non-canonical glutamine transporter, SLC38A1, was found to be overexpressed in MDS stem cells; was associated with clinical responses to telaglenastat/AZA and predictive of worse prognosis in a large MDS cohort. These data demonstrate the safety and efficacy of a combined metabolic and epigenetic approach in MDS.

Complex I inhibitor of oxidative phosphorylation in advanced solid tumors and acute myeloid leukemia: phase I trials.

Although targeting oxidative phosphorylation (OXPHOS) is a rational anticancer strategy, clinical benefit with OXPHOS inhibitors has yet to be achieved. Here we advanced IACS-010759, a highly potent and selective small-molecule complex I inhibitor, into two dose-escalation phase I trials in patients with relapsed/refractory acute myeloid leukemia (NCT02882321, n = 17) and advanced solid tumors (NCT03291938, n = 23). The primary endpoints were safety, tolerability, maximum tolerated dose and recommended phase 2 dose (RP2D) of IACS-010759. The PK, PD, and preliminary antitumor activities of IACS-010759 in patients were also evaluated as secondary endpoints in both clinical trials. IACS-010759 had a narrow therapeutic index with emergent dose-limiting toxicities, including elevated blood lactate and neurotoxicity, which obstructed efforts to maintain target exposure. Consequently no RP2D was established, only modest target inhibition and limited antitumor activity were observed at tolerated doses, and both trials were discontinued. Reverse translational studies in mice demonstrated that IACS-010759 induced behavioral and physiological changes indicative of peripheral neuropathy, which were minimized with the coadministration of a histone deacetylase 6 inhibitor. Additional studies are needed to elucidate the association between OXPHOS inhibition and neurotoxicity, and caution is warranted in the continued development of complex I inhibitors as antitumor agents.

Landscape of MicroRNA Regulatory Network Architecture and Functional Rerouting in Cancer.

Somatic mutations are a major source of cancer development, and many driver mutations have been identified in protein coding regions. However, the function of mutations located in miRNA and their target binding sites throughout the human genome remains largely unknown. Here, we built detailed cancer-specific miRNA regulatory networks across 30 cancer types to systematically analyze the effect of mutations in miRNAs and their target sites in 3' untranslated region (3' UTR), coding sequence (CDS), and 5' UTR regions. A total of 3,518,261 mutations from 9,819 samples were mapped to miRNA-gene interactions (mGI). Mutations in miRNAs showed a mutually exclusive pattern with mutations in their target genes in almost all cancer types. A linear regression method identified 148 candidate driver mutations that can significantly perturb miRNA regulatory networks. Driver mutations in 3'UTRs played their roles by altering RNA binding energy and the expression of target genes. Finally, mutated driver gene targets in 3' UTRs were significantly downregulated in cancer and functioned as tumor suppressors during cancer progression, suggesting potential miRNA candidates with significant clinical implications. A user-friendly, open-access web portal (mGI-map) was developed to facilitate further use of this data resource. Together, these results will facilitate novel noncoding biomarker identification and therapeutic drug design targeting the miRNA regulatory networks. SIGNIFICANCE: A detailed miRNA-gene interaction map reveals extensive miRNA-mediated gene regulatory networks with mutation-induced perturbations across multiple cancers, serving as a resource for noncoding biomarker discovery and drug development.

Circulating metabolites associated with tumor hypoxia and early response to treatment in bevacizumab-refractory glioblastoma after combined bevacizumab and evofosfamide.

Glioblastomas (GBM) are the most common and aggressive form of primary malignant brain tumor in the adult population, and, despite modern therapies, patients often develop recurrent disease, and the disease remains incurable with median survival below 2 years. Resistance to bevacizumab is driven by hypoxia in the tumor and evofosfamide is a hypoxia-activated prodrug, which we tested in a phase 2, dual center (University of Texas Health Science Center in San Antonio and Dana Farber Cancer Institute) clinical trial after bevacizumab failure. Tumor hypoxic volume was quantified by 18F-misonidazole PET. To identify circulating metabolic biomarkers of tumor hypoxia in patients, we used a high-resolution liquid chromatography-mass spectrometry-based approach to profile blood metabolites and their specific enantiomeric forms using untargeted approaches. Moreover, to evaluate early response to treatment, we characterized changes in circulating metabolite levels during treatment with combined bevacizumab and evofosfamide in recurrent GBM after bevacizumab failure. Gamma aminobutyric acid, and glutamic acid as well as its enantiomeric form D-glutamic acid all inversely correlated with tumor hypoxia. Intermediates of the serine synthesis pathway, which is known to be modulated by hypoxia, also correlated with tumor hypoxia (phosphoserine and serine). Moreover, following treatment, lactic acid was modulated by treatment, likely in response to a hypoxia mediated modulation of oxidative vs glycolytic metabolism. In summary, although our results require further validation in larger patients' cohorts, we have identified candidate metabolic biomarkers that could evaluate the extent of tumor hypoxia and predict the benefit of combined bevacizumab and evofosfamide treatment in GBM following bevacizumab failure.

Metabolomics-based phenotypic screens for evaluation of drug synergy via direct-infusion mass spectrometry.

Drugs used in combination can synergize to increase efficacy, decrease toxicity, and prevent drug resistance. While conventional high-throughput screens that rely on univariate data are incredibly valuable to identify promising drug candidates, phenotypic screening methodologies could be beneficial to provide deep insight into the molecular response of drug combination with a likelihood of improved clinical outcomes. We developed a high-content metabolomics drug screening platform using stable isotope-tracer direct-infusion mass spectrometry that informs an algorithm to determine synergy from multivariate phenomics data. Using a cancer drug library, we validated the drug screening, integrating isotope-enriched metabolomics data and computational data mining, on a panel of prostate cell lines and verified the synergy between CB-839 and docetaxel both in vitro (three-dimensional model) and in vivo. The proposed unbiased metabolomics screening platform can be used to rapidly generate phenotype-informed datasets and quantify synergy for combinatorial drug discovery.

Inhibition of mitochondrial complex I reverses NOTCH1-driven metabolic reprogramming in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is commonly driven by activating mutations in NOTCH1 that facilitate glutamine oxidation. Here we identify oxidative phosphorylation (OxPhos) as a critical pathway for leukemia cell survival and demonstrate a direct relationship between NOTCH1, elevated OxPhos gene expression, and acquired chemoresistance in pre-leukemic and leukemic models. Disrupting OxPhos with IACS-010759, an inhibitor of mitochondrial complex I, causes potent growth inhibition through induction of metabolic shut-down and redox imbalance in NOTCH1-mutated and less so in NOTCH1-wt T-ALL cells. Mechanistically, inhibition of OxPhos induces a metabolic reprogramming into glutaminolysis. We show that pharmacological blockade of OxPhos combined with inducible knock-down of glutaminase, the key glutamine enzyme, confers synthetic lethality in mice harboring NOTCH1-mutated T-ALL. We leverage on this synthetic lethal interaction to demonstrate that IACS-010759 in combination with chemotherapy containing L-asparaginase, an enzyme that uncovers the glutamine dependency of leukemic cells, causes reduced glutaminolysis and profound tumor reduction in pre-clinical models of human T-ALL. In summary, this metabolic dependency of T-ALL on OxPhos provides a rational therapeutic target.

Activation of RAS/MAPK pathway confers MCL-1 mediated acquired resistance to BCL-2 inhibitor venetoclax in acute myeloid leukemia.

Despite high initial response rates, acute myeloid leukemia (AML) treated with the BCL-2-selective inhibitor venetoclax (VEN) alone or in combinations commonly acquires resistance. We performed gene/protein expression, metabolomic and methylation analyses of isogenic AML cell lines sensitive or resistant to VEN, and identified the activation of RAS/MAPK pathway, leading to increased stability and higher levels of MCL-1 protein, as a major acquired mechanism of VEN resistance. MCL-1 sustained survival and maintained mitochondrial respiration in VEN-RE cells, which had impaired electron transport chain (ETC) complex II activity, and MCL-1 silencing or pharmacologic inhibition restored VEN sensitivity. In support of the importance of RAS/MAPK activation, we found by single-cell DNA sequencing rapid clonal selection of RAS-mutated clones in AML patients treated with VEN-containing regimens. In summary, these findings establish RAS/MAPK/MCL-1 and mitochondrial fitness as key survival mechanisms of VEN-RE AML and provide the rationale for combinatorial strategies effectively targeting these pathways.

Automated Trimethyl Sulfonium Hydroxide Derivatization Method for High-Throughput Fatty Acid Profiling by Gas Chromatography-Mass Spectrometry.

Fatty acid profiling on gas chromatography-mass spectrometry (GC-MS) platforms is typically performed offline by manually derivatizing and analyzing small batches of samples. A GC-MS system with a fully integrated robotic autosampler can significantly improve sample handling, standardize data collection, and reduce the total hands-on time required for sample analysis. In this study, we report an optimized high-throughput GC-MS-based methodology that utilizes trimethyl sulfonium hydroxide (TMSH) as a derivatization reagent to convert fatty acids into fatty acid methyl esters. An automated online derivatization method was developed, in which the robotic autosampler derivatizes each sample individually and injects it into the GC-MS system in a high-throughput manner. This study investigated the robustness of automated TMSH derivatization by comparing fatty acid standards and lipid extracts, derivatized manually in batches and online automatically from four biological matrices. Automated derivatization improved reproducibility in 19 of 33 fatty acid standards, with nearly half of the 33 confirmed fatty acids in biological samples demonstrating improved reproducibility when compared to manually derivatized samples. In summary, we show that the online TMSH-based derivatization methodology is ideal for high-throughput fatty acid analysis, allowing rapid and efficient fatty acid profiling, with reduced sample handling, faster data acquisition, and, ultimately, improved data reproducibility.

The Combined Treatment With the FLT3-Inhibitor AC220 and the Complex I Inhibitor IACS-010759 Synergistically Depletes Wt- and FLT3-Mutated Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a high mortality rate and relapse risk. Although progress on the genetic and molecular understanding of this disease has been made, the standard of care has changed minimally for the past 40 years and the five-year survival rate remains poor, warranting new treatment strategies. Here, we applied a two-step screening platform consisting of a primary cell viability screening and a secondary metabolomics-based phenotypic screening to find synergistic drug combinations to treat AML. A novel synergy between the oxidative phosphorylation inhibitor IACS-010759 and the FMS-like tyrosine kinase 3 (FLT3) inhibitor AC220 (quizartinib) was discovered in AML and then validated by ATP bioluminescence and apoptosis assays. In-depth stable isotope tracer metabolic flux analysis revealed that IACS-010759 and AC220 synergistically reduced glucose and glutamine enrichment in glycolysis and the TCA cycle, leading to impaired energy production and de novo nucleotide biosynthesis. In summary, we identified a novel drug combination, AC220 and IACS-010759, which synergistically inhibits cell growth in AML cells due to a major disruption of cell metabolism, regardless of FLT3 mutation status.

Gilteritinib Inhibits Glutamine Uptake and Utilization in FLT3-ITD-Positive AML.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy associated with frequent relapse and poor overall survival. The tyrosine kinase inhibitor gilteritinib is approved for the treatment of relapse/refractory AML with FLT3 mutations, yet its mechanism of action is not completely understood. Here, we sought to identify additional therapeutic targets that can be exploited to enhance gilteritinib's antileukemic effect. Based on unbiased transcriptomic analyses, we identified the glutamine transporter SNAT1 (SLC38A1) as a novel target of gilteritinib that leads to impaired glutamine uptake and utilization within leukemic cells. Using metabolomics and metabolic flux analyses, we found that gilteritinib decreased glutamine metabolism through the TCA cycle and cellular levels of the oncometabolite 2-hydroxyglutarate. In addition, gilteritinib treatment was associated with decreased ATP production and glutathione synthesis and increased reactive oxygen species, resulting in cellular senescence. Finally, we found that the glutaminase inhibitor CB-839 enhanced antileukemic effect of gilteritinib in ex vivo studies using human primary FLT3-ITD-positive AML cells harboring mutations in the enzyme isocitrate dehydrogenase, which catalyzes the oxidative decarboxylation of isocitrate, producing α-ketoglutarate. Collectively, this work has identified a previously unrecognized, gilteritinib-sensitive metabolic pathway downstream of SLC38A1 that causes decreased glutaminolysis and disruption of redox homeostasis. These findings provide a rationale for the development and therapeutic exploration of targeted combinatorial treatment strategies for this subset of relapse/refractory AML.

Novel Strategy for Untargeted Chiral Metabolomics using Liquid Chromatography-High Resolution Tandem Mass Spectrometry.

Stereospecific recognition of metabolites plays a significant role in the detection of potential disease biomarkers thereby providing new insights in diagnosis and prognosis. D-Hdroxy/amino acids are recognized as potential biomarkers in several metabolic disorders. Despite continuous advances in metabolomics technologies, the simultaneous measurement of different classes of enantiomeric metabolites in a single analytical run remains challenging. Here, we develop a novel strategy for untargeted chiral metabolomics of hydroxy/amine groups (-OH/-NH2) containing metabolites, including all hydroxy acids (HAs) and amino acids (AAs), by chiral derivatization coupled with liquid chromatography-high resolution tandem mass spectrometry (LC-HR-MS/MS). Diacetyl-tartaric anhydride (DATAN) was used for the simultaneous derivatization of-OH/-NH2 containing metabolites as well as the resulting diastereomers, and all the derivatized metabolites were resolved in a single analytical run. Data independent MS/MS acquisition (DIA) was applied to positively identify DATAN-labeled metabolites based on reagent specific diagnostic fragment ions. We discriminated chiral from achiral metabolites based on the reversal of elution order of D and L isomers derivatized with the enantiomeric pair (±) of DATAN in an untargeted manner. Using the developed strategy, a library of 301 standards that consisted of 214 chiral and 87 achiral metabolites were separated and detected in a single analytical run. This approach was then applied to investigate the enantioselective metabolic profile of the bone marrow (BM) and peripheral blood (PB) plasma samples from patients with acute myeloid leukemia (AML) at diagnosis and following completion of the induction phase of chemotherapeutic treatment. The sensitivity and selectivity of the developed method enabled the detection of trace levels of the D-enantiomer of HAs and AAs in primary plasma patient samples. Several of these metabolites were significantly altered in response to chemotherapy. The developed LC-HR-MS method entails a valuable step forward in chiral metabolomics.

The low molecular weight protein tyrosine phosphatase promotes adipogenesis and subcutaneous adipocyte hypertrophy.

Obesity is a major contributing factor to the pathogenesis of Type 2 diabetes. Multiple human genetics studies suggest that high activity of the low molecular weight protein tyrosine phosphatase (LMPTP) promotes metabolic syndrome in obesity. We reported that LMPTP is a critical promoter of insulin resistance in obesity by regulating liver insulin receptor signaling and that inhibition of LMPTP reverses obesity-associated diabetes in mice. Since LMPTP is expressed in adipose tissue but little is known about its function, here we examined the role of LMPTP in adipocyte biology. Using conditional knockout mice, we found that selective deletion of LMPTP in adipocytes impaired obesity-induced subcutaneous adipocyte hypertrophy. We assessed the role of LMPTP in adipogenesis in vitro, and found that LMPTP deletion or knockdown substantially impaired differentiation of primary preadipocytes and 3T3-L1 cells into adipocytes, respectively. Inhibition of LMPTP in 3T3-L1 preadipocytes also reduced adipogenesis and expression of proadipogenic transcription factors peroxisome proliferator activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha. Inhibition of LMPTP increased basal phosphorylation of platelet-derived growth factor receptor alpha (PDGFRα) on activation motif residue Y849 in 3T3-L1, resulting in increased activation of the mitogen-associated protein kinases p38 and c-Jun N-terminal kinase and increased PPARγ phosphorylation on inhibitory residue S82. Analysis of the metabolome of differentiating 3T3-L1 cells suggested that LMPTP inhibition decreased cell glucose utilization while enhancing mitochondrial respiration and nucleotide synthesis. In summary, we report a novel role for LMPTP as a key driver of adipocyte differentiation via control of PDGFRα signaling.

Predicting and Quantifying Antagonistic Effects of Natural Compounds Given with Chemotherapeutic Agents: Applications for High-Throughput Screening.

Natural products have been used for centuries to treat various human ailments. In recent decades, multi-drug combinations that utilize natural products to synergistically enhance the therapeutic effects of cancer drugs have been identified and have shown success in improving treatment outcomes. While drug synergy research is a burgeoning field, there are disagreements on the definitions and mathematical parameters that prevent the standardization and proper usage of the terms synergy, antagonism, and additivity. This contributes to the relatively small amount of data on the antagonistic effects of natural products on cancer drugs that can diminish their therapeutic efficacy and prevent cancer regression. The ability of natural products to potentially degrade or reverse the molecular activity of cancer therapeutics represents an important but highly under-emphasized area of research that is often overlooked in both pre-clinical and clinical studies. This review aims to evaluate the body of work surrounding the antagonistic interactions between natural products and cancer therapeutics and highlight applications for high-throughput screening (HTS) and deep learning techniques for the identification of natural products that antagonize cancer drug efficacy.

Stable Isotope Tracing Metabolomics to Investigate the Metabolic Activity of Bioactive Compounds for Cancer Prevention and Treatment.

A major hallmark of cancer is the metabolic reprogramming of cancer cells to fuel tumor growth and proliferation. Various plant-derived bioactive compounds efficiently target the metabolic vulnerabilities of cancer cells and exhibit potential as emerging therapeutic agents. Due to their safety and common use as dietary components, they are also ideal for cancer prevention. However, to render their use as efficient as possible, the mechanism of action of these phytochemicals needs to be well characterized. Stable isotope tracing is an essential technology to study the molecular mechanisms by which nutraceuticals modulate and target cancer metabolism. The use of positionally labeled tracers as exogenous nutrients and the monitoring of their downstream metabolites labeling patterns enable the analysis of the specific metabolic pathway activity, via the relative production and consumption of the labeled metabolites. Although stable isotope tracing metabolomics is a powerful tool to investigate the molecular activity of bioactive compounds as well as to design synergistic nutraceutical combinations, this methodology is still underutilized. This review aims to investigate the research efforts and potentials surrounding the use of stable isotope tracing metabolomics to examine the metabolic alterations mediated by bioactive compounds in cancer.

Enzyme-mediated depletion of serum l-Met abrogates prostate cancer growth via multiple mechanisms without evidence of systemic toxicity.

Extensive studies in prostate cancer and other malignancies have revealed that l-methionine (l-Met) and its metabolites play a critical role in tumorigenesis. Preclinical and clinical studies have demonstrated that systemic restriction of serum l-Met, either via partial dietary restriction or with bacterial l-Met-degrading enzymes exerts potent antitumor effects. However, administration of bacterial l-Met-degrading enzymes has not proven practical for human therapy because of problems with immunogenicity. As the human genome does not encode l-Met-degrading enzymes, we engineered the human cystathionine-γ-lyase (hMGL-4.0) to catalyze the selective degradation of l-Met. At therapeutically relevant dosing, hMGL-4.0 reduces serum l-Met levels to >75% for >72 h and significantly inhibits the growth of multiple prostate cancer allografts/xenografts without weight loss or toxicity. We demonstrate that in vitro, hMGL-4.0 causes tumor cell death, associated with increased reactive oxygen species, S-adenosyl-methionine depletion, global hypomethylation, induction of autophagy, and robust poly(ADP-ribose) polymerase (PARP) cleavage indicative of DNA damage and apoptosis.

Identification of a synergistic combination of dimethylaminoparthenolide and shikonin alters metabolism and inhibits proliferation of pediatric precursor-B cell acute lymphoblastic leukemia.

Exploiting metabolic vulnerabilities of cancer cells with nontoxic, plant derived compounds constitutes a novel strategy for both chemoprevention and treatment. A high-throughput screening approach was used to evaluate a library of natural products to determine the most synergistic combination in precursor-B cell acute lymphoblast leukemia. Dimethylaminoparthenolide and shikonin effectively inhibited proliferation resulting in cell death in primary and immortalized leukemia cells, while having negligible effects on normal cells. Dimethylaminoparthenolide and shikonin have been shown separately to inhibit cell survival and proliferative signaling and activate tumor suppressors and proapoptotic pathways. Untargeted metabolomics and metabolic flux analysis with stable isotopically labeled glucose and glutamine exhibited a global shift in metabolism following treatment. Pathway analysis indicated significant differences in amino acid, antioxidant, tricarboxylic acid cycle, and nucleotide metabolism. Together, dimethylaminoparthenolide and shikonin reduced the shunting of glycolytic intermediates into the pentose phosphate pathway for biosynthetic purposes. Similarly, the incorporation of glutamine and glutamine-derived metabolites into purine and pyrimidine synthesis was inhibited by the combination of dimethylaminoparthenolide and shikonin, effectively impeding biosynthetic pathways critical for leukemia cell survival. This approach demonstrates that a synergistic pair of compounds with malignant cell specificity can effectively target metabolic pathways crucial to leukemia cell proliferation and induce apoptosis.