Modulation of Cell Surface Protein Free Thiols: A Potential Novel Mechanism of Action of the Sesquiterpene Lactone Parthenolide

There has been much interest in targeting intracellular redox pathways as a therapeutic approach for cancer. Given recent data to suggest that the redox status of extracellular protein thiol groups (i.e. exofacial thiols) effects cell behavior, we hypothesized that redox active anti-cancer agents would modulate exofacial protein thiols.To test this hypothesis, we used the sesquiterpene lactone parthenolide, a known anti-cancer agent. Using flow cytometry, and western blotting to label free thiols with Alexa Fluor 633 C(5) maleimide dye and N-(biotinoyl)-N-(iodoacetyl) ethylendiamine (BIAM), respectively, we show that parthenolide decreases the level of free exofacial thiols on Granta mantle lymphoma cells. In addition, we used immuno-precipitation techniques to identify the central redox regulator thioredoxin, as one of the surface protein thiol targets modified by parthenolide. To examine the functional role of parthenolide induced surface protein thiol modification, we pretreated Granta cells with cell impermeable glutathione (GSH), prior to exposure to parthenolide, and showed that GSH pretreatment; (a) inhibited the interaction of parthenolide with exofacial thiols; (b) inhibited parthenolide mediated activation of JNK and inhibition of NFkappaB, two well established mechanisms of parthenolide activity and; (c) blocked the cytotoxic activity of parthenolide. That GSH had no effect on the parthenolide induced generation of intracellular reactive oxygen species supports the fact that GSH had no effect on intracellular redox. Together these data support the likelihood that GSH inhibits the effect of parthenolide on JNK, NFkappaB and cell death through its direct inhibition of parthenolide's modulation of exofacial thiols.Based on these data, we postulate that one component of parthenolide's anti-lymphoma activity derives from its ability to modify the redox state of critical exofacial thiols. Further, we propose that cancer cell exofacial thiols may be important and novel targets for therapy

A small-molecule inhibitor of the aberrant transcription factor CBFβ-SMMHC delays leukemia in mice

This is the author’s version of the work. It is posted here by permission of the AAAS for personal use, not for redistribution. The definitive version was published in Science on 2015 February 13; 347(6223): 779–784, DOI: 10.1126/science.aaa0314.Acute myeloid leukemia (AML) is the most common form of adult leukemia. The transcription factor fusion CBFβ-SMMHC (core binding factor β and the smooth-muscle myosin heavy chain), expressed in AML with the chromosome inversion inv(16)(p13q22), outcompetes wild-type CBFβ for binding to the transcription factor RUNX1, deregulates RUNX1 activity in hematopoiesis, and induces AML. Current inv(16) AML treatment with nonselective cytotoxic chemotherapy results in a good initial response but limited long-term survival. Here, we report the development of a protein-protein interaction inhibitor, AI-10-49, that selectively binds to CBFβ-SMMHC and disrupts its binding to RUNX1. AI-10-49 restores RUNX1 transcriptional activity, displays favorable pharmacokinetics, and delays leukemia progression in mice. Treatment of primary inv(16) AML patient blasts with AI-10-49 triggers selective cell death. These data suggest that direct inhibition of the oncogenic CBFβ-SMMHC fusion protein may be an effective therapeutic approach for inv(16) AML, and they provide support for transcription factor targeted therapy in other cancers

Pharmacologic inhibition of the Menin-MLL interaction blocks progression of MLL leukemia in vivo

Chromosomal translocations affecting mixed lineage leukemia gene (MLL) result in acute leukemias resistant to therapy. The leukemogenic activity of MLL fusion proteins is dependent on their interaction with menin, providing basis for therapeutic intervention. Here we report the development of highly potent and orally bioavailable small-molecule inhibitors of the menin-MLL interaction, MI-463 and MI-503, and show their profound effects in MLL leukemia cells and substantial survival benefit in mouse models of MLL leukemia. Finally, we demonstrate the efficacy of these compounds in primary samples derived from MLL leukemia patients. Overall, we demonstrate that pharmacologic inhibition of the menin-MLL interaction represents an effective treatment for MLL leukemias in vivo and provide advanced molecular scaffold for clinical lead identification

Technical Aspects of Flow Cytometry-based Measurable Residual Disease Quantification in Acute Myeloid Leukemia: Experience of the European LeukemiaNet MRD Working Party

Measurable residual disease (MRD) quantified by multiparameter flow cytometry (MFC) is a strong and independent prognostic factor in acute myeloid leukemia (AML). However, several technical factors may affect the final read-out of the assay. Experts from the MRD Working Party of the European LeukemiaNet evaluated which aspects are crucial for accurate MFC-MRD measurement. Here, we report on the agreement, obtained via a combination of a cross-sectional questionnaire, live discussions, and a Delphi poll. The recommendations consist of several key issues from bone marrow sampling to final laboratory reporting to ensure quality and reproducibility of results. Furthermore, the experiences were tested by comparing two 8-color MRD panels in multiple laboratories. The results presented here underscore the feasibility and the utility of a harmonized theoretical and practical MFC-MRD assessment and are a next step toward further harmonization

Endemic Venezuelan Equine Encephalitis in Northern Peru

Since Venezuelan equine encephalitis virus (VEEV) was isolated in Peru in 1942, >70 isolates have been obtained from mosquitoes, humans, and sylvatic mammals primarily in the Amazon region. To investigate genetic relationships among the Peru VEEV isolates and between the Peru isolates and other VEEV strains, a fragment of the PE2 gene was amplified and analyzed by single-stranded conformation polymorphism. Representatives of seven genotypes underwent sequencing and phylogenetic analysis. The results identified four VEE complex lineages that cocirculate in the Amazon region: subtypes ID (Panama and Colombia/Venezuela genotypes), IIIC, and a new, proposed subtype IIID, which was isolated from a febrile human, mosquitoes, and spiny rats. Both ID lineages and the IIID subtype are associated with febrile human illness. Most of the subtype ID isolates belonged to the Panama genotype, but the Colombia/Venezuela genotype, which is phylogenetically related to epizootic strains, also continues to circulate in the Amazon basin

Updates on radiotherapy-immunotherapy combinations: Proceedings of 6(th) annual ImmunoRad conference

Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference

Clonal Hematopoiesis Before, During, and After Human Spaceflight.

Clonal hematopoiesis (CH) occurs when blood cells harboring an advantageous mutation propagate faster than others. These mutations confer a risk for hematological cancers and cardiovascular disease. Here, we analyze CH in blood samples from a pair of twin astronauts over 4 years in bulk and fractionated cell populations using a targeted CH panel, linked-read whole-genome sequencing, and deep RNA sequencing. We show CH with distinct mutational profiles and increasing allelic fraction that includes a high-risk, TET2 clone in one subject and two DNMT3A mutations on distinct alleles in the other twin. These astronauts exhibit CH almost two decades prior to the mean age at which it is typically detected and show larger shifts in clone size than age-matched controls or radiotherapy patients, based on a longitudinal cohort of 157 cancer patients. As such, longitudinal monitoring of CH may serve as an important metric for overall cancer and cardiovascular risk in astronauts