Rational Drug Design of Targeted and Enzyme-Cleavable Vitamin E Analogs as a Neoadjuvant to Chemotherapy: In Vitro and In Vivo Evaluation on Reduction of the Cardiotoxicity Side Effect of Doxorubicin.

Traditional drug design focuses on specific biological targets where specific receptors or biomarkers are overexpressed by cancer cells. Cancer cells circumvent the interventions by activating survival pathways and/or downregulating cell death pathways for their survival. A priori activation of apoptosis pathways of tumor (AAAPT) is a novel tumor-sensitizing technology that sensitizes tumor cells that are not responding well to the current treatments by targeting specific survival pathways involved in the desensitization of tumor cells and tries to revive them selectively in cancer cells, sparing normal cells. Several vitamin E derivatives (AMP-001, AMP-002, AMP-003, and AMP-004) were synthesized, characterized, and studied for their anti-tumorigenic properties and their synergistic potential with the standard chemotherapy doxorubicin in various cancer cells including brain cancer stem cells in vitro. Preliminary studies revealed that AAAPT drugs (a) reduced the invasive potential of brain tumor stem cells, (b) synergized with Federal Drug Application-approved doxorubicin, and (c) enhanced the therapeutic index of doxorubicin in the triple-negative breast cancer tumor rat model, preserving the ventricular function compared to cardiotoxic doxorubicin alone at therapeutic dose. The AAAPT approach has the advantage of inhibiting survival pathways and activating cell death pathways selectively in cancer cells by using targeting, linkers cleavable by tumor-specific Cathepsin B, and PEGylation technology to enhance the bioavailability. We propose AAAPT drugs as a neoadjuvant to chemotherapy and not as stand-alone therapy, which is shown to be effective in expanding the therapeutic index of doxorubicin and making it work at lower doses.

Early Noninvasive Metabolic Biomarkers of Mutant IDH Inhibition in Glioma.

Approximately 80% of low-grade glioma (LGGs) harbor mutant isocitrate dehydrogenase 1/2 (IDH1/2) driver mutations leading to accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). Thus, inhibition of mutant IDH is considered a potential therapeutic target. Several mutant IDH inhibitors are currently in clinical trials, including AG-881 and BAY-1436032. However, to date, early detection of response remains a challenge. In this study we used high resolution 1H magnetic resonance spectroscopy (1H-MRS) to identify early noninvasive MR (Magnetic Resonance)-detectable metabolic biomarkers of response to mutant IDH inhibition. In vivo 1H-MRS was performed on mice orthotopically-implanted with either genetically engineered (U87IDHmut) or patient-derived (BT257 and SF10417) mutant IDH1 cells. Treatment with either AG-881 or BAY-1436032 induced a significant reduction in 2-HG. Moreover, both inhibitors led to a significant early and sustained increase in glutamate and the sum of glutamate and glutamine (GLX) in all three models. A transient early increase in N-acetylaspartate (NAA) was also observed. Importantly, all models demonstrated enhanced animal survival following both treatments and the metabolic alterations were observed prior to any detectable differences in tumor volume between control and treated tumors. Our study therefore identifies potential translatable early metabolic biomarkers of drug delivery, mutant IDH inhibition and glioma response to treatment with emerging clinically relevant therapies.

Mutant IDH1 gliomas downregulate phosphocholine and phosphoethanolamine synthesis in a 2-hydroxyglutarate-dependent manner.

BACKGROUND: Magnetic resonance spectroscopy (MRS) studies have identified elevated levels of the phospholipid precursor phosphocholine (PC) and phosphoethanolamine (PE) as metabolic hallmarks of cancer. Unusually, however, PC and PE levels are reduced in mutant isocitrate dehydrogenase 1 (IDHmut) gliomas that produce the oncometabolite 2-hydroxyglutarate (2-HG) relative to wild-type IDH1 (IDHwt) gliomas. The goal of this study was to determine the molecular mechanism underlying this unusual metabolic reprogramming in IDHmut gliomas. METHODS: Steady-state PC and PE were quantified using 31P-MRS. To quantify de novo PC and PE synthesis, we used 13C-MRS and measured flux to 13C-PC and 13C-PE in cells incubated with [1,2-13C]-choline and [1,2-13C]-ethanolamine. The activities of choline kinase (CK) and ethanolamine kinase (EK), the enzymes responsible for PC and PE synthesis, were quantified using 31P-MR-based assays. To interrogate the role of 2-HG, we examined IDHwt cells incubated with 2-HG and, conversely, IDHmut cells treated with the IDHmut inhibitor AGI-5198. To examine the role of hypoxia-inducible factor 1-α (HIF-1α), we silenced HIF-1α using RNA interference. To confirm our findings in vivo and in the clinic, we studied IDHwt and IDHmut orthotopic tumor xenografts and glioma patient biopsies. RESULTS: De novo synthesis of PC and PE was reduced in IDHmut cells relative to IDHwt. Concomitantly, CK activity and EK activity were reduced in IDHmut cells. Pharmacological manipulation of 2-HG levels established that 2-HG was responsible for reduced CK activity, EK activity, PC and PE. 2-HG has previously been reported to stabilize levels of HIF-1α, a known regulator of CK activity. Silencing HIF-1α in IDHmut cells restored CK activity, EK activity, PC and PE to IDHwt levels. Our findings were recapitulated in IDHmut orthotopic tumor xenografts and, most importantly, in IDHmut patient biopsies, validating our findings in vivo and in the clinic. CONCLUSIONS: This study identifies, to our knowledge for the first time, a direct role for 2-HG in the downregulation of CK and EK activity, and thereby, PC and PE synthesis in IDHmut gliomas. These results highlight the unusual reprogramming of phospholipid metabolism in IDHmut gliomas and have implications for the identification of MRS-detectable metabolic biomarkers associated with 2-HG status.

2-Hydroxyglutarate-Mediated Autophagy of the Endoplasmic Reticulum Leads to an Unusual Downregulation of Phospholipid Biosynthesis in Mutant IDH1 Gliomas.

Tumor metabolism is reprogrammed to meet the demands of proliferating cancer cells. In particular, cancer cells upregulate synthesis of the membrane phospholipids phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdE) in order to allow for rapid membrane turnover. Nonetheless, we show here that, in mutant isocitrate dehydrogenase 1 (IDHmut) gliomas, which produce the oncometabolite 2-hydroxyglutarate (2-HG), PtdCho and PtdE biosynthesis is downregulated and results in lower levels of both phospholipids when compared with wild-type IDH1 cells. 2-HG inhibited collagen-4-prolyl hydroxylase activity, leading to accumulation of misfolded procollagen-IV in the endoplasmic reticulum (ER) of both genetically engineered and patient-derived IDHmut glioma models. The resulting ER stress triggered increased expression of FAM134b, which mediated autophagic degradation of the ER (ER-phagy) and a reduction in the ER area. Because the ER is the site of phospholipid synthesis, ER-phagy led to reduced PtdCho and PtdE biosynthesis. Inhibition of ER-phagy via pharmacological or molecular approaches restored phospholipid biosynthesis in IDHmut glioma cells, triggered apoptotic cell death, inhibited tumor growth, and prolonged the survival of orthotopic IDHmut glioma-bearing mice, pointing to a potential therapeutic opportunity. Glioma patient biopsies also exhibited increased ER-phagy and downregulation of PtdCho and PtdE levels in IDHmut samples compared with wild-type, clinically validating our observations. Collectively, this study provides detailed and clinically relevant insights into the functional link between oncometabolite-driven ER-phagy and phospholipid biosynthesis in IDHmut gliomas.Significance: Downregulation of phospholipid biosynthesis via ER-phagy is essential for proliferation and clonogenicity of mutant IDH1 gliomas, a finding with immediate therapeutic implications. Cancer Res; 78(9); 2290-304. ©2018 AACR.

The JAK2/STAT3 inhibitor pacritinib effectively inhibits patient-derived GBM brain tumor initiating cells in vitro and when used in combination with temozolomide increases survival in an orthotopic xenograft model.

PURPOSE: The prognosis for patients diagnosed with glioblastoma multiforme (GBM) remains dismal, with current treatment prolonging survival only modestly. As such, there remains a strong need for novel therapeutic strategies. The janus kinase (JAK)2/signal transducer and activator of transcription (STAT)3 pathway regulates many cellular processes in GBM, including survival, proliferation, invasion, anti-apoptosis, and immune evasion. Here, we evaluated the preclinical efficacy of pacritinib, a novel compound targeting JAK2, using a collection of diverse patient-derived brain tumor initiating cells (BTICs). EXPERIMENTAL DESIGN: The effects of pacritinib on BTIC viability and sphere forming capacity were evaluated in vitro using the alamarBlue and neurosphere assays, respectively. On-target inhibition of JAK2/STAT3 signaling was investigated using western blotting. The efficacy of pacritinib was tested in vivo in pharmacokinetic analyses, liver microsome analyses, and Kaplan-Meier survival studies. RESULTS: In vitro, pacritinib decreased BTIC viability and sphere forming potential at low micromolar doses and demonstrated on-target inhibition of STAT3 signaling. Additionally, pacritinib was found to improve the response to temozolomide (TMZ) in TMZ-resistant BTICs. In vivo, systemic treatment with pacritinib demonstrated blood-brain barrier penetration and led to improved overall median survival in combination with TMZ, in mice orthotopically xenografted with an aggressive recurrent GBM BTIC culture. CONCLUSION: This preclinical study demonstrates the efficacy of pacritinib and supports the feasibility of testing pacritinib for the treatment of GBM, in combination with the standard of care TMZ.