Inhibition of Geranylgeranyl Diphosphate Synthase is a Novel Therapeutic Strategy for Pancreatic Ductal Adenocarcinoma

Rab proteins play an essential role in regulating intracellular membrane trafficking processes. Rab activity is dependent upon geranylgeranylation, a post-translational modification that involves the addition of 20-carbon isoprenoid chains via the enzyme geranylgeranyl transferase (GGTase) II. We have focused on the development of inhibitors against geranylgeranyl diphosphate synthase (GGDPS), which generates the isoprenoid donor (GGPP), as anti-Rab agents. Pancreatic ductal adenocarcinoma (PDAC) is characterized by abnormal mucin production and these mucins play important roles in tumor development, metastasis and chemo-resistance. We hypothesized that GGDPS inhibitor (GGDPSi) treatment would induce PDAC cell death by disrupting mucin trafficking, thereby inducing the unfolded protein response pathway (UPR) and apoptosis. To this end, we evaluated the effects of RAM2061, a potent GGDPSi, against PDAC. Our studies revealed that GGDPSi treatment activates the UPR and triggers apoptosis in a variety of human and mouse PDAC cell lines. Furthermore, GGDPSi treatment was found to disrupt the intracellular trafficking of key mucins such as MUC1. These effects could be recapitulated by incubation with a specific GGTase II inhibitor, but not a GGTase I inhibitor, consistent with the effect being dependent on disruption of Rab-mediated activities. In addition, siRNA-mediated knockdown of GGDPS induces upregulation of UPR markers and disrupts MUC1 trafficking in PDAC cells. Experiments in two mouse models of PDAC demonstrated that GGDPSi treatment significantly slows tumor growth. Collectively, these data support further development of GGDPSi therapy as a novel strategy for the treatment of PDAC

Targeting the Isoprenoid Biosynthetic Pathway in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy for which there is currently no cure. While treatment options for MM have expanded over the last two decades, all patients will eventually become resistant to current therapies. Thus, there is an urgent need for novel therapeutic strategies to treat MM. The isoprenoid biosynthetic pathway (IBP) is responsible for the post-translational modification of proteins belonging to the Ras small GTPase superfamily, such as Ras, Rho and Rab family members. Given the important roles these GTPase proteins play in various cellular processes, there is significant interest in the development of inhibitors that disturb their prenylation and consequently their activity in MM cells. Numerous preclinical studies have demonstrated that IBP inhibitors have anti-MM effects, including the induction of apoptosis in MM cells and inhibition of osteoclast activity. Some IBP inhibitors have made their way into the clinic. For instance, nitrogenous bisphosphonates are routinely prescribed for the management MM bone disease. Other IBP inhibitors, including statins and farnesyltransferase inhibitors, have been evaluated in clinical trials for MM, while there is substantial preclinical investigation into geranylgeranyl diphosphate synthase inhibitors. Here we discuss recent advances in the development of IBP inhibitors, assess their mechanism of action and evaluate their potential as anti-MM agents

Targeting the Isoprenoid Biosynthetic Pathway in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy for which there is currently no cure. While treatment options for MM have expanded over the last two decades, all patients will eventually become resistant to current therapies. Thus, there is an urgent need for novel therapeutic strategies to treat MM. The isoprenoid biosynthetic pathway (IBP) is responsible for the post-translational modification of proteins belonging to the Ras small GTPase superfamily, such as Ras, Rho and Rab family members. Given the important roles these GTPase proteins play in various cellular processes, there is significant interest in the development of inhibitors that disturb their prenylation and consequently their activity in MM cells. Numerous preclinical studies have demonstrated that IBP inhibitors have anti-MM effects, including the induction of apoptosis in MM cells and inhibition of osteoclast activity. Some IBP inhibitors have made their way into the clinic. For instance, nitrogenous bisphosphonates are routinely prescribed for the management MM bone disease. Other IBP inhibitors, including statins and farnesyltransferase inhibitors, have been evaluated in clinical trials for MM, while there is substantial preclinical investigation into geranylgeranyl diphosphate synthase inhibitors. Here we discuss recent advances in the development of IBP inhibitors, assess their mechanism of action and evaluate their potential as anti-MM agents

Simultaneous Quantitation of Isoprenoid Pyrophosphates in Plasma and Cancer Cells Using LC-MS/MS

Isoprenoids (IsoP) are an important class of molecules involved in many different cellular processes including cholesterol synthesis. We have developed a sensitive and specific LC-MS/MS method for the quantitation of three key IsoPs in bio-matrices, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP). LC-MS/MS analysis was performed using a Nexera UPLC System connected to a LCMS-8060 (Shimadzu Scientific Instruments, Columbia, MD) with a dual ion source. The electrospray ionization source was operated in the negative MRM mode. The chromatographic separation and detection of analytes was achieved on a reversed phase ACCQ-TAG Ultra C18 (1.7 µm, 100 mm × 2.1 mm I.D.) column. The mobile phase consisted of (1) a 10 mM ammonium carbonate with 0.1% ammonium hydroxide in water, and (2) a 0.1% ammonium hydroxide in acetonitrile/methanol (75/25). The flow rate was set to 0.25 mL/min in a gradient condition. The limit of quantification was 0.04 ng/mL for all analytes with a correlation coefficient (r2) of 0.998 or better and a total run time of 12 min. The inter- and intra-day accuracy (85⁻115%) precision (<15%), and recovery (40⁻90%) values met the acceptance criteria. The validated method was successfully applied to quantitate basal concentrations of GPP, FPP and GGPP in human plasma and in cultured cancer cell lines. Our LC-MS/MS method may be used for IsoP quantification in different bio-fluids and to further investigate the role of these compounds in various physiological processes

Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma

<div><p>DNA methyltransferase 3A (DNMT3A) is an enzyme involved in DNA methylation that is frequently mutated in human hematologic malignancies. We have previously shown that inactivation of Dnmt3a in hematopoietic cells results in chronic lymphocytic leukemia in mice. Here we show that 12% of Dnmt3a-deficient mice develop CD8+ mature peripheral T cell lymphomas (PTCL) and 29% of mice are affected by both diseases. 10% of <i>Dnmt3a</i>^<i>+/-</i> mice develop lymphomas, suggesting that Dnmt3a is a haploinsufficient tumor suppressor in PTCL. DNA methylation was deregulated genome-wide with 10-fold more hypo- than hypermethylated promoters and enhancers, demonstrating that hypomethylation is a major event in the development of PTCL. Hypomethylated promoters were enriched for binding sites of transcription factors AML1, NF-κB and OCT1, implying the transcription factors potential involvement in Dnmt3a-associated methylation. Whereas 71 hypomethylated genes showed an increased expression in PTCL, only 3 hypermethylated genes were silenced, suggesting that cancer-specific hypomethylation has broader effects on the transcriptome of cancer cells than hypermethylation. Interestingly, transcriptomes of <i>Dnmt3a</i>^<i>+/-</i> and <i>Dnmt3a</i>^<i>Δ/Δ</i> lymphomas were largely conserved and significantly overlapped with those of human tumors. Importantly, we observed downregulation of tumor suppressor p53 in <i>Dnmt3a</i>^<i>+/-</i> and <i>Dnmt3a</i>^<i>Δ/Δ</i> lymphomas as well as in pre-tumor thymocytes from 9 months old but not 6 weeks old <i>Dnmt3a</i>^<i>+/-</i> tumor-free mice, suggesting that p53 downregulation is chronologically an intermediate event in tumorigenesis. Decrease in p53 is likely an important event in tumorigenesis because its overexpression inhibited proliferation in mouse PTCL cell lines, suggesting that low levels of p53 are important for tumor maintenance. Altogether, our data link the haploinsufficient tumor suppressor function of Dnmt3a in the prevention of mouse mature CD8+ PTCL indirectly to a <i>bona fide</i> tumor suppressor of T cell malignancies p53.</p></div

Promoter and gene-body hypomethylation is present throughout the genome of <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCLs.

<p>Circos plot of DMRS annotated to promoters and gene bodies in <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL relative to <i>wild-type</i> CD8+ cells. DMRs aligning to promoter (outer circle) and gene body (inner circles) are displayed in relation to its chromosomal position in the mouse genome. Hypomethylated DMRS are indicated by yellow lines and hypermethylated DMRS are indicated by blue lines.</p

Jdp2 is hypomethylated and overexpressed in human and mouse PTCL.

<p>(A) COBRA analysis of mouse Jdp2 promoter methylation in three independent <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL samples. Undigested (U) and digested (D) fragments correspond to unmethylated and methylated DNA, respectively. Control PCR fragments generated from fully methylated mouse genomic DNA that is undigested (M) or digested (CpG) are shown. (B) Normalized gene expression of Jdp2 in mouse CD8+ control, <i>Dnmt3a</i>^<i>+/-</i> PTCL, and <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL samples by qRT-PCR. Data presented are the average of two independent experiments. Error bars show standard deviation and an asterisk (*) denotes a p<0.05 (student t-test). (C) Bisulfite sequencing of the JDP2 promoter in normal human CD3+ T cells and in two independent human PTCL samples. Circles represent individual CpGs within the promoter. Black and white areas denote the relative portion of methylated and un-methylated sequence reads at a CpG, respectively. (D) Normalized gene expression of JDP2 in normal human CD3+ T cells and human PTCL samples, by qRT-PCR. Data presented are the average of two independent experiments. Error bars show standard deviation and an asterisk (*) denotes a p<0.05 (student t-test).</p

A majority of promoters are methylated and inactive in normal mouse CD8+ T cells.

<p>(A) CpG methylation in <i>wild-type</i> CD8+ cells, as determined by WGBS. Individual CpGs were placed into quartiles based on percent methylation (0–25%, 26–50%, 51–75%, and 76–100%). (B) Percent methylation shown by quartiles for core promoter regions (-300bp to +150bp relative to the TSS) in <i>wild-type</i> CD8+ cells. Methylation percentages for all CpGs across the 450bp region were averaged to give a mean methylation value for each gene’s core promoter. (C) A heat map displaying methylation status of 21,712 promoters in <i>wild-type</i> CD8+ as determined by WGBS. Methylation percentage for individual CpGs were annotated to the promoter regions −300bp to +150bp relative to the transcription start site (TSS). Methylation percentages for all CpGs across the 450bp region were averaged to give a mean methylation value for each gene promoter. Lowly methylated promoters are shown in yellow and highly methylated promoters in blue (D) Heat map presentation of gene-matched promoter methylation (as analyzed in panel C) and corresponding transcriptional expression (averaged FPKM values) in <i>wild-type</i> CD8+ cells, as determined by WGBS and RNA-seq for 15,732 genes. Highly expressed genes are denoted in red and lowly expressed genes are denoted in green. (E) Ingenuity Pathway analysis (IPA) of highly expressed genes (FPKM ≥ 10) in <i>wild-type</i> CD8+ cells. The top subcategories obtained in Physiological System, Development and Functions are displayed (P<0.05, for all subcategories).</p