A clinical survey of mosaic single nucleotide variants in disease-causing genes detected by exome sequencing.

BACKGROUND: Although mosaic variation has been known to cause disease for decades, high-throughput sequencing technologies with the analytical sensitivity to consistently detect variants at reduced allelic fractions have only recently emerged as routine clinical diagnostic tests. To date, few systematic analyses of mosaic variants detected by diagnostic exome sequencing for diverse clinical indications have been performed. METHODS: To investigate the frequency, type, allelic fraction, and phenotypic consequences of clinically relevant somatic mosaic single nucleotide variants (SNVs) and characteristics of the corresponding genes, we retrospectively queried reported mosaic variants from a cohort of ~ 12,000 samples submitted for clinical exome sequencing (ES) at Baylor Genetics. RESULTS: We found 120 mosaic variants involving 107 genes, including 80 mosaic SNVs in proband samples and 40 in parental/grandparental samples. Average mosaic alternate allele fraction (AAF) detected in autosomes and in X-linked disease genes in females was 18.2% compared with 34.8% in X-linked disease genes in males. Of these mosaic variants, 74 variants (61.7%) were classified as pathogenic or likely pathogenic and 46 (38.3%) as variants of uncertain significance. Mosaic variants occurred in disease genes associated with autosomal dominant (AD) or AD/autosomal recessive (AR) (67/120, 55.8%), X-linked (33/120, 27.5%), AD/somatic (10/120, 8.3%), and AR (8/120, 6.7%) inheritance. Of note, 1.7% (2/120) of variants were found in genes in which only somatic events have been described. Nine genes had recurrent mosaic events in unrelated individuals which accounted for 18.3% (22/120) of all detected mosaic variants in this study. The proband group was enriched for mosaicism affecting Ras signaling pathway genes. CONCLUSIONS: In sum, an estimated 1.5% of all molecular diagnoses made in this cohort could be attributed to a mosaic variant detected in the proband, while parental mosaicism was identified in 0.3% of families analyzed. As ES design favors breadth over depth of coverage, this estimate of the prevalence of mosaic variants likely represents an underestimate of the total number of clinically relevant mosaic variants in our cohort.

Reversal of the glycolytic phenotype of primary effusion lymphoma cells by combined targeting of cellular metabolism and PI3K/Akt/ mTOR signaling.

PEL is a B-cell non-Hodgkin lymphoma, occurring predominantly as a lymphomatous effusion in body cavities, characterized by aggressive clinical course, with no standard therapy. Based on previous reports that PEL cells display a Warburg phenotype, we hypothesized that the highly hypoxic environment in which they grow in vivo makes them more reliant on glycolysis, and more vulnerable to drugs targeting this pathway. We established here that indeed PEL cells in hypoxia are more sensitive to glycolysis inhibition. Furthermore, since PI3K/Akt/mTOR has been proposed as a drug target in PEL, we ascertained that pathway-specific inhibitors, namely the dual PI3K and mTOR inhibitor, PF-04691502, and the Akt inhibitor, Akti 1/2, display improved cytotoxicity to PEL cells in hypoxic conditions. Unexpectedly, we found that these drugs reduce lactate production/extracellular acidification rate, and, in combination with the glycolysis inhibitor 2-deoxyglucose (2-DG), they shift PEL cells metabolism from aerobic glycolysis towards oxidative respiration. Moreover, the associations possess strong synergistic cytotoxicity towards PEL cells, and thus may reduce adverse reaction in vivo, while displaying very low toxicity to normal lymphocytes. Finally, we showed that the association of 2-DG and PF-04691502 maintains its cytotoxic and proapoptotic effect also in PEL cells co-cultured with human primary mesothelial cells, a condition known to mimic the in vivo environment and to exert a protective and pro-survival action. All together, these results provide a compelling rationale for the clinical development of new therapies for the treatment of PEL, based on combined targeting of glycolytic metabolism and constitutively activated signaling pathways.

Clinical application of whole-exome sequencing across clinical indications.

PURPOSE: We report the diagnostic yield of whole-exome sequencing (WES) in 3,040 consecutive cases at a single clinical laboratory. METHODS: WES was performed for many different clinical indications and included the proband plus two or more family members in 76% of cases. RESULTS: The overall diagnostic yield of WES was 28.8%. The diagnostic yield was 23.6% in proband-only cases and 31.0% when three family members were analyzed. The highest yield was for patients who had disorders involving hearing (55%, N = 11), vision (47%, N = 60), the skeletal muscle system (40%, N = 43), the skeletal system (39%, N = 54), multiple congenital anomalies (36%, N = 729), skin (32%, N = 31), the central nervous system (31%, N = 1,082), and the cardiovascular system (28%, N = 54). Of 2,091 cases in which secondary findings were analyzed for 56 American College of Medical Genetics and Genomics-recommended genes, 6.2% (N = 129) had reportable pathogenic variants. In addition to cases with a definitive diagnosis, in 24.2% of cases a candidate gene was reported that may later be reclassified as being associated with a definitive diagnosis. CONCLUSION: Our experience with our first 3,040 WES cases suggests that analysis of trios significantly improves the diagnostic yield compared with proband-only testing for genetically heterogeneous disorders and facilitates identification of novel candidate genes.Genet Med 18 7, 696-704.

Pathogenic and likely pathogenic variant prevalence among the first 10,000 patients referred for next-generation cancer panel testing.

PURPOSE: Germ-line testing for panels of cancer genes using next-generation sequencing is becoming more common in clinical care. We report our experience as a clinical laboratory testing both well-established, high-risk cancer genes (e.g., BRCA1/2, MLH1, MSH2) as well as more recently identified cancer genes (e.g., PALB2, BRIP1), many of which have increased but less well-defined penetrance. METHODS: Clinical genetic testing was performed on over 10,000 consecutive cases referred for evaluation of germ-line cancer genes, and results were analyzed for frequency of pathogenic or likely pathogenic variants, and were stratified by testing panel, gene, and clinical history. RESULTS: Overall, a molecular diagnosis was made in 9.0% of patients tested, with the highest yield in the Lynch syndrome/colorectal cancer panel. In patients with breast, ovarian, or colon/stomach cancer, positive yields were 9.7, 13.4, and 14.8%, respectively. Approximately half of the pathogenic variants identified in patients with breast or ovarian cancer were in genes other than BRCA1/2. CONCLUSION: The high frequency of positive results in a wide range of cancer genes, including those of high penetrance and with clinical care guidelines, underscores both the genetic heterogeneity of hereditary cancer and the usefulness of multigene panels over genetic tests of one or two genes.Genet Med 18 8, 823-832.

Further supporting evidence for the SATB2-associated syndrome found through whole exome sequencing.

The SATB2-associated syndrome (SAS) was recently proposed as a clinically recognizable syndrome that results from deleterious alterations of the SATB2 gene in humans. Although interstitial deletions at 2q33 encompassing SATB2, either alone or contiguously with other genes, have been reported before, there is limited literature regarding intragenic mutations of this gene and the resulting phenotype. We describe five patients in whom whole exome sequencing identified five unique de novo mutations in the SATB2 gene (one splice site, one frameshift, and three nonsense mutations). The five patients had overlapping features that support the characteristic features of the SAS: intellectual disability with limited speech development and craniofacial abnormalities including cleft palate, dysmorphic features, and dental abnormalities. Furthermore, Patient 1 also had features not previously described that represent an expansion of the phenotype. Osteopenia was seen in two of the patients, suggesting that this finding could be added to the list of distinctive findings. We provide supporting evidence that analysis for deletions or point mutations in SATB2 should be considered in children with intellectual disability and severely impaired speech, cleft or high palate, teeth abnormalities, and osteopenia.

Assessing copy number from exome sequencing and exome array CGH based on CNV spectrum in a large clinical cohort.

PURPOSE: Detection of copy-number variation (CNV) is important for investigating many genetic disorders. Testing a large clinical cohort by array comparative genomic hybridization provides a deep perspective on the spectrum of pathogenic CNV. In this context, we describe a bioinformatics approach to extract CNV information from whole-exome sequencing and demonstrate its utility in clinical testing. METHODS: Exon-focused arrays and whole-genome chromosomal microarray analysis were used to test 14,228 and 14,000 individuals, respectively. Based on these results, we developed an algorithm to detect deletions/duplications in whole-exome sequencing data and a novel whole-exome array. RESULTS: In the exon array cohort, we observed a positive detection rate of 2.4% (25 duplications, 318 deletions), of which 39% involved one or two exons. Chromosomal microarray analysis identified 3,345 CNVs affecting single genes (18%). We demonstrate that our whole-exome sequencing algorithm resolves CNVs of three or more exons. CONCLUSION: These results demonstrate the clinical utility of single-exon resolution in CNV assays. Our whole-exome sequencing algorithm approaches this resolution but is complemented by a whole-exome array to unambiguously identify intragenic CNVs and single-exon changes. These data illustrate the next advancements in CNV analysis through whole-exome sequencing and whole-exome array.Genet Med 17 8, 623-629.

The protein kinase Akt/PKB regulates both prelamin A degradation and Lmna gene expression.

The serine/threonine kinase Akt/PKB is a major signaling hub integrating metabolic, survival, growth, and cell cycle regulatory signals. The definition of the phospho-motif cipher driving phosphorylation by Akt led to the identification of hundreds of putative substrates, and it is therefore pivotal to identify those whose phosphorylation by Akt is of consequence to biological processes. The Lmna gene products lamin A/C and the lamin A precursor prelamin A are type V intermediate filament proteins forming a filamentous meshwork, the lamina, underneath the inner nuclear membrane, for nuclear envelope structures organization and interphase chromatin anchoring. In our previous work, we reported that A-type lamins are phosphorylated by Akt at S301 and S404 in physiological conditions and are therefore bona fide substrates of Akt. We report here that Akt phosphorylation at S404 targets the precursor prelamin A for degradation. We further demonstrate that Akt also regulates Lmna transcription. Our study unveils a previously unknown function of Akt in the control of prelamin A stability and expression. Moreover, given the large number of diseases related to prelamin A, our findings represent a further important step bridging basic A-type lamin physiology to therapeutic approaches for lamin A-linked disorders.

Structure-based selectivity optimization of piperidine-pteridine derivatives as potent Leishmania pteridine reductase inhibitors.

The upregulation of pteridine reductase (PTR1) is a major contributor to antifolate drug resistance in Leishmania spp., as it provides a salvage pathway that bypasses dihydrofolate reductase (DHFR) inhibition. The structure-based optimization of the PTR1 inhibitor methyl-1-[4-(2,4-diaminopteridin-6-ylmethylamino)benzoyl]piperidine-4-carboxylate (1) led to the synthesis of a focused compound library which showed significantly improved selectivity for the parasite's folate-dependent enzyme. When used in combination with pyrimethamine, a DHFR inhibitor, a synergistic effect was observed for compound 5b. This work represents a step forward in the identification of effective antileishmania agents.

ON 01910.Na is selectively cytotoxic for chronic lymphocytic leukemia cells through a dual mechanism of action involving PI3K/AKT inhibition and induction of oxidative stress.

PURPOSE: Chronic lymphocytic leukemia (CLL), a malignancy of mature B cells, is incurable with chemotherapy. Signals from the microenvironment support leukemic cell survival and proliferation and may confer chemotherapy resistance. ON 01910.Na (Rigosertib), a multikinase phosphoinositide 3-kinase (PI3K) inhibitor, is entering phase III trials for myelodysplastic syndrome. Our aim was to analyze the efficacy of ON 01910.Na against CLL cells in vitro and investigate the molecular effects of this drug on tumor biology. EXPERIMENTAL DESIGN: Cytotoxicity of ON 01910.Na against CLL cells from 34 patients was determined in vitro with flow cytometry of cells stained with Annexin V and CD19. Global gene expression profiling on Affymetrix microarrays, flow cytometry, Western blotting, and cocultures with stroma cells were used to delineate ON 01910.Na mechanism of action. RESULTS: ON 01910.Na induced apoptosis in CLL B cells without significant toxicity against T cells or normal B cells. ON 01910.Na was equally active against leukemic cells associated with a more aggressive disease course [immunoglobulin heavy-chain variable region unmutated, adverse cytogenetics] than against cells without these features. Gene expression profiling revealed two main mechanisms of action: PI3K/AKT inhibition and induction of ROS that resulted in an oxidative stress response through activating protein 1 (AP-1), c-jun-NH(2)-terminal kinase, and ATF3 culminating in the upregulation of NOXA. ROS scavengers and shRNA mediated knockdown of ATF3- and NOXA-protected cells from drug-induced apoptosis. ON 01910.Na also abrogated the prosurvival effect of follicular dendritic cells on CLL cells and reduced SDF-1-induced migration of leukemic cells. CONCLUSIONS: These data support the clinical development of ON 01910.Na in CLL.

Activation of CD44, a receptor for extracellular matrix components, protects chronic lymphocytic leukemia cells from spontaneous and drug induced apoptosis through MCL-1.

Survival of chronic lymphocytic leukemia (CLL) cells in vivo is supported by the tissue microenvironment, which includes components of the extracellular matrix. Interactions between tumor cells and the extracellular matrix are in part mediated by CD44, whose principal ligand is hyaluronic acid. Here, we show that CD44 is more highly expressed on CLL cells of the clinically more progressive immunglobulin heavy chain variable gene (IGHV)-unmutated subtype than on cells of the IGHV-mutated type. Engagement of CD44 activated the phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen activated protein kinase (MAPK)/ERK pathways and increased myeloid cell leukemia sequence 1 (MCL-1) protein expression. Consistent with the induction of these anti-apoptotic mechanisms, CD44 protected CLL cells from spontaneous and fludarabine-induced apoptosis. Obatoclax, an antagonist of MCL-1, blocked the pro-survival effect of CD44. In addition, obatoclax synergized with fludarabine to induce apoptosis of CLL cells. In conclusion, components of the extracellular matrix may provide survival signals to CLL cells through engagement of CD44. Inhibition of MCL-1 is a promising strategy to reduce the anti-apoptotic effect of the microenvironment on CLL cells.

The lymph node microenvironment promotes B-cell receptor signaling, NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL), an incurable malignancy of mature B lymphocytes, involves blood, bone marrow, and secondary lymphoid organs such as the lymph nodes (LN). A role of the tissue microenvironment in the pathogenesis of CLL is hypothesized based on in vitro observations, but its contribution in vivo remains ill-defined. To elucidate the effects of tumor-host interactions in vivo, we purified tumor cells from 24 treatment-naive patients. Samples were obtained concurrently from blood, bone marrow, and/or LN and analyzed by gene expression profiling. We identified the LN as a key site in CLL pathogenesis. CLL cells in the LN showed up-regulation of gene signatures, indicating B-cell receptor (BCR) and nuclear factor-κB activation. Consistent with antigen-dependent BCR signaling and canonical nuclear factor-κB activation, we detected phosphorylation of SYK and IκBα, respectively. Expression of BCR target genes was stronger in clinically more aggressive CLL, indicating more effective BCR signaling in this subtype in vivo. Tumor proliferation, quantified by the expression of the E2F and c-MYC target genes and verified with Ki67 staining by flow cytometry, was highest in the LN and was correlated with clinical disease progression. These data identify the disruption of tumor microenvironment interactions and the inhibition of BCR signaling as promising therapeutic strategies in CLL. This study is registered at http://clinicaltrials.gov as NCT00019370.

Lipidomic analysis of bloodstream and procyclic form Trypanosoma brucei.

The biological membranes of Trypanosoma brucei contain a complex array of phospholipids that are synthesized de novo from precursors obtained either directly from the host, or as catabolised endocytosed lipids. This paper describes the use of nanoflow electrospray tandem mass spectrometry and high resolution mass spectrometry in both positive and negative ion modes, allowing the identification of approximately 500 individual molecular phospholipids species from total lipid extracts of cultured bloodstream and procyclic form T. brucei. Various molecular species of all of the major subclasses of glycerophospholipids were identified including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol as well as phosphatidic acid, phosphatidylglycerol and cardolipin, and the sphingolipids sphingomyelin, inositol phosphoceramide and ethanolamine phosphoceramide. The lipidomic data obtained in this study will aid future biochemical phenotyping of either genetically or chemically manipulated commonly used bloodstream and procyclic strains of Trypanosoma brucei. Hopefully this will allow a greater understanding of the bizarre world of lipids in this important human pathogen.

The Kennedy pathway--De novo synthesis of phosphatidylethanolamine and phosphatidylcholine.

The glycerophospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) account for greater than 50% of the total phospholipid species in eukaryotic membranes and thus play major roles in the structure and function of those membranes. In most eukaryotic cells, PC and PE are synthesized by an aminoalcoholphosphotransferase reaction, which uses sn-1,2-diradylglycerol and either CDP-choline or CDP-ethanolamine, respectively. This is the last step in a biosynthetic pathway known as the Kennedy pathway, so named after Eugene Kennedy who elucidated it over 50 years ago. This review will cover various aspects of the Kennedy pathway including: each of the biosynthetic steps, the functions and roles of the phospholipid products PC and PE, and how the Kennedy pathway has the potential of being a chemotherapeutic target against cancer and various infectious diseases.

Lenalidomide-induced upregulation of CD80 on tumor cells correlates with T-cell activation, the rapid onset of a cytokine release syndrome and leukemic cell clearance in chronic lymphocytic leukemia.

BACKGROUND: In chronic lymphocytic leukemia lenalidomide causes striking immune activation, possibly leading to clearance of tumor cells. We conducted this study to investigate the mechanism of action of lenalidomide and the basis for its unique toxicities in chronic lymphocytic leukemia. DESIGN AND METHODS: Patients with relapsed chronic lymphocytic leukemia were treated with lenalidomide 20 mg (n=10) or 10 mg (n=8) daily for 3 weeks on a 6-week cycle. Correlative studies assessed expression of co-stimulatory molecules on tumor cells, T-cell activation, cytokine levels, and changes in lymphocyte subsets. RESULTS: Lenalidomide upregulated the co-stimulatory molecule CD80 on chronic lymphocytic leukemia and mantle cell lymphoma cells but not on normal peripheral blood B cells in vitro. T-cell activation was apparent in chronic lymphocytic leukemia, weak in mantle cell lymphoma, but absent in normal peripheral blood mononuclear cells and correlated with the upregulation of CD80 on B cells. Strong CD80 upregulation and T-cell activation predicted more severe side effects, manifesting in 83% of patients as a cytokine release syndrome within 8-72 h after the first dose of lenalidomide. Serum levels of various cytokines, including tumor necrosis factor-alpha, increased during treatment. CD80 upregulation on tumor cells correlated with rapid clearance of leukemic cells from the peripheral blood. In contrast, neither the severity of the cytokine release syndrome nor the degree of T-cell activation in vitro correlated with clinical response. CONCLUSIONS: Upregulation of CD80 on tumor cells and T-cell activation correlate with unique toxicities of lenalidomide in chronic lymphocytic leukemia. However, T-cell activation appears to be dispensable for the drug's anti-tumor effects. This provides a rationale for combinations of lenalidomide with fludarabine or alemtuzumab.

The ethanolamine branch of the Kennedy pathway is essential in the bloodstream form of Trypanosoma brucei.

Phosphatidylethanolamine (GPEtn), a major phospholipid component of trypanosome membranes, is synthesized de novo from ethanolamine through the Kennedy pathway. Here the composition of the GPEtn molecular species in the bloodstream form of Trypanosoma brucei is determined, along with new insights into phospholipid metabolism, by in vitro and in vivo characterization of a key enzyme of the Kennedy pathway, the cytosolic ethanolamine-phosphate cytidylyltransferase (TbECT). Gene knockout indicates that TbECT is essential for growth and survival, thus highlighting the importance of the Kennedy pathway for the pathogenic stage of the African trypanosome. Phosphatiylserine decarboxylation, a potential salvage pathway, does not appear to be active in cultured bloodstream form T. brucei, and it is not upregulated even when the Kennedy pathway is disrupted. In vivo metabolic labelling and phospholipid composition analysis by ESI-MS/MS of the knockout cells confirmed a significant decrease in GPEtn species, as well as changes in the relative abundance of other phospholipid species. Reduction in GPEtn levels had a profound influence on the morphology of the mutants and it compromised mitochondrial structure and function, as well as glycosylphosphatidylinositol anchor biosynthesis. TbECT is therefore genetically validated as a potential drug target against the African trypanosome.

Role of perforin-mediated cell apoptosis in murine models of infusion-induced bone marrow failure.

OBJECTIVE: To investigate the role of perforin-mediated cell apoptosis in murine models of immune-mediated bone marrow (BM) failure. MATERIALS AND METHODS: We compared C57BL/6J (B6) mice carrying a perforin gene deletion (Prf(-/-)) with wild-type (WT) controls for cellular composition in lymphohematopoietic tissues. Lymph node (LN) cells from Prf(-/-) mice were coincubated with BM cells from B10-H2(b)/LilMcdJ (C.B10) mice in an apoptosis assay in vitro. We then infused Prf(-/-) and WT B6 LN cells into sublethally irradiated C.B10 and CByB6F1 recipients with mismatches at the minor and major histocompatibility loci, respectively, in order to induce BM failure. Cellular composition was analyzed by flow cytometry. RESULTS: Prf(-/-) mice showed normal lymphoid cell composition, but Prf(-/-) LN cells had reduced ability to induce C.B10 BM cell apoptosis in vitro. Infusion of 5 to 10 x 10(6) Prf(-/-) LN cells produced obvious BM failure in C.B10 and CByB6F1 recipients; pancytopenia and BM hypocellularity were only slightly less severe than those caused by infusion of 5 x 10(6) WT B6 LN cells. Infused Prf(-/-) LN cells showed less T-cell expansion, normal T-cell activation, and higher proportions of T cells expressing gamma-interferon, tissue necrosis factor-alpha, and Fas ligand CD178, in comparison to infused WT B6 LN cells. Fas expression was equally high in residual BM cells in recipient of both Prf(-/-) and B6 LN cells. CONCLUSION: Perforin deficiency alters T-cell expansion but upregulates T-cell Fas ligand expression. Perforin-mediated cell death appears to play a minor role in mouse models of immune-mediated BM failure.

Biochemical characterization of the initial steps of the Kennedy pathway in Trypanosoma brucei: the ethanolamine and choline kinases.

Ethanolamine and choline are major components of the trypanosome membrane phospholipids, in the form of GPEtn (phosphatidylethanolamine) [corrected] and GPCho (phosphatidylcholine) [corrected] . Ethanolamine is also found as an integral component of the GPI (glycosylphosphatidylinositol) anchor that is required for membrane attachment of cell-surface proteins, most notably the variant-surface glycoproteins. The de novo synthesis of GPEtn and GPCho starts with the generation of phosphoethanolamine and phosphocholine by ethanolamine and choline kinases via the Kennedy pathway. Database mining revealed two putative C/EKs (choline/ethanolamine kinases) in the Trypanosoma brucei genome, which were cloned, overexpressed, purified and characterized. TbEK1 (T. brucei ethanolamine kinase 1) was shown to be catalytically active as an ethanolamine-specific kinase, i.e. it had no choline kinase activity. The K(m) values for ethanolamine and ATP were found to be 18.4+/-0.9 and 219+/-29 microM respectively. TbC/EK2 (T. brucei choline/ethanolamine kinase 2), on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a K(m) 80 times lower than that of ethanolamine. The K(m) values for choline, ethanolamine and ATP were 31.4+/-2.6 microM, 2.56+/-0.31 mM and 20.6+/-1.96 microM respectively. Further substrate specificity analysis revealed that both TbEK1 and TbC/EK2 were able to tolerate various modifications at the amino group, with the exception of a quaternary amine for TbEK1 (choline) and a primary amine for TbC/EK2 (ethanolamine). Both enzymes recognized analogues with substituents on C-2, but substitutions on C-1 and elongations of the carbon chain were not well tolerated.

Noxa mediates bortezomib induced apoptosis in both sensitive and intrinsically resistant mantle cell lymphoma cells and this effect is independent of constitutive activity of the AKT and NF-kappaB pathways.

Bortezomib is more active against mantle cell lymphoma (MCL) than against most other lymphoma subtypes. Nevertheless, up to half of patients with MCL have bortezomib resistant disease. Factors contributing to intrinsic resistance to bortezomib have not been determined. Here we used a panel of eight bortezomib sensitive (median IC(50) 5.9 nM) and three relatively bortezomib resistant cell lines (median IC(50) 12.9 nM) to investigate differences in tumor biology that could determine sensitivity to bortezomib. Bortezomib effectively inhibited high baseline proteasome activity and induced a comparable degree of proteasome inhibition in both sensitive and resistant cells. At 10 nM, bortezomib induced the proapoptotic BH3-only protein Noxa in sensitive but not resistant cells. At higher concentrations of bortezomib, however, Noxa was also upregulated in resistant cells and this effect was sufficient to induce apoptosis. Silencing of Noxa with siRNA rescued these cells from apoptosis, arguing against a defect in Noxa regulation or function as the basis of bortezomib resistance. Bortezomib was equally effective against cells with high and low constitutive NF-kappaB signaling. Also, sensitive and resistant MCL cell lines showed comparable activation of the AKT pathway. We conclude that bortezomib can overcome classic mechanisms of resistance to apoptosis and that determinants of bortezomib sensitivity in MCL are due to differences in signaling or stress pathways upstream of Noxa.

Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development.

Pteridine reductase (PTR1) is essential for salvage of pterins by parasitic trypanosomatids and is a target for the development of improved therapies. To identify inhibitors of Leishmania major and Trypanosoma cruzi PTR1, we combined a rapid-screening strategy using a folate-based library with structure-based design. Assays were carried out against folate-dependent enzymes including PTR1, dihydrofolate reductase (DHFR), and thymidylate synthase. Affinity profiling determined selectivity and specificity of a series of quinoxaline and 2,4-diaminopteridine derivatives, and nine compounds showed greater activity against parasite enzymes compared with human enzymes. Compound 6a displayed a K(i) of 100 nM toward LmPTR1, and the crystal structure of the LmPTR1:NADPH:6a ternary complex revealed a substrate-like binding mode distinct from that previously observed for similar compounds. A second round of design, synthesis, and assay produced a compound (6b) with a significantly improved K(i) (37 nM) against LmPTR1, and the structure of this complex was also determined. Biological evaluation of selected inhibitors was performed against the extracellular forms of T. cruzi and L. major, both wild-type and overexpressing PTR1 lines, as a model for PTR1-driven antifolate drug resistance and the intracellular form of T. cruzi. An additive profile was observed when PTR1 inhibitors were used in combination with known DHFR inhibitors, and a reduction in toxicity of treatment was observed with respect to administration of a DHFR inhibitor alone. The successful combination of antifolates targeting two enzymes indicates high potential for such an approach in the development of previously undescribed antiparasitic drugs.

Decreased TCR zeta-chain expression in T cells from patients with acquired aplastic anaemia.

In aplastic anaemia, T cells have a central role in the pathophysiology of bone marrow destruction. This study showed that T cells from patients with aplastic anaemia expressed decreased T-cell receptor (TCR) zeta-chain protein and mRNA levels compared to healthy controls. Patients with decreased TCR zeta-chain showed an abnormal response in intracellular calcium following stimulation through the TCR. We also observed an altered pattern of the transcription factors CREMalpha and Elf-1 that are implicated in zeta-chain transcription. We concluded that TCR zeta-chain expression was decreased in the majority of patients with aplastic anaemia, regardless of disease activity or treatment status.