Characterizing Protein Dynamics of Protein-Ligand Interactions by Hydrogen-Deuterium Exchange Mass Spectrometry

The study of protein-ligand and protein-protein interactions is of paramount importance to the understanding of their biological function. Whereas this area of research has been largely dominated by conventional structural biology techniques, such as NMR and X-ray crystallography, an emerging methodology that relies on the implementation of hydrogen deuterium exchange (HDX) powered by MS-based analysis holds the potential to greatly expand on our ability to probe the protein dynamics of fundamental biological processes. In this work, the entire HDX workflow for site-specific analysis of protein dynamics was integrated onto a concerted microfluidic device and applied to the interrogation of the dynamic changes that accompany protein-ligand interactions. This application is described for two model systems: the binding of glutathione (GSH) by Glutathione-S-Transferase (GST), and the binding of three novel salicylic acid-based inhibitors of Signal Transducer and Activator of Transcription 3 (STAT3) to its SH2 domain. This work extends the application of time-resolved electrospray ionization mass spectrometry (TRESI-MS) HDX to the study of protein ligand interaction dynamics and ligand-binding site mapping

Recent developments in immunotherapy of acute myeloid leukemia

The advent of new immunotherapeutic agents in clinical practice has revolutionized cancer treatment in the past decade, both in oncology and hematology. The transfer of the immunotherapeutic concepts to the treatment of acute myeloid leukemia (AML) is hampered by various characteristics of the disease, including non-leukemia-restricted target antigen expression profile, low endogenous immune responses, and intrinsic resistance mechanisms of the leukemic blasts against immune responses. However, considerable progress has been made in this field in the past few years. Within this manuscript, we review the recent developments and the current status of the five currently most prominent immunotherapeutic concepts: (1) antibody-drug conjugates, (2) T cell-recruiting antibody constructs, (3) chimeric antigen receptor (CAR) T cells, (4) checkpoint inhibitors, and (5) dendritic cell vaccination. We focus on the clinical data that has been published so far, both for newly diagnosed and refractory/relapsed AML, but omitting immunotherapeutic concepts in conjunction with hematopoietic stem cell transplantation. Besides, we have included important clinical trials that are currently running or have recently been completed but are still lacking full publication of their results. While each of the concepts has its particular merits and inherent problems, the field of immunotherapy of AML seems to have taken some significant steps forward. Results of currently running trials will reveal the direction of further development including approaches combining two or more of these concepts

Abstract 2010: Charactering the interactomes of the Myc family of oncogenes

Abstract The Myc family of transcription factors, c-Myc, N-Myc and L-Myc, are known to be deregulated in a large variety of cancers. Mechanisms responsible for the deregulation of the activity of Myc family members in cancer are not well understood. A number of protein-protein interactions and post-translational modifications have been suggested to promote the oncogenic activity of Myc. Traditional biochemical approaches have not been successful at mapping the interactors of Myc family members due to their tight association with chromatin and the labile nature of Myc proteins. Mapping the protein-protein interactions that support the aberrant activity of Myc family of oncoproteins in cancer cells is of high interest, particularly in a more natural context in xenograft models in vivo. A new mass spectrometry-based technique, BioID-MS, which relies on proximity-based biotin labeling, has recently emerged as a key advance for the characterization of hard-to-detect protein-protein interactions in living cells. Herein, we are reporting a new application of the BioID-MS technique for the characterization of c-Myc interactors in human cell line in vivo, in mouse tumor xenografts. Using the in vivo BioID assay, we were able to identify more than 30 known and validated c-Myc interactors, some of which include the components of the STAGA complex and SWI/SNF chromatin remodelling complex. We were further able to identify more than 100 novel high-confidence c-Myc interactors, which include components of the DNA repair and replication machinery, general transcription and elongation factors, and the co-regulator of transcription-like DNA helicase protein chromodomain 8 (CHD8). Using ENCODE ChIP-seq datasets we were able to map some of the high-confidence interactors to coincident binding sites with c-Myc throughout the genome. This provided further credibility that the newly identified putative interactors could co-occupy sites on chromatin with c-Myc and could be functionally important for activity. Furthermore, we validated the Myc-CHD8 interaction using a number of approaches, including yeast two hybrid and proximity-based ligation assays. These findings suggest that the BioID-MS technique can be used to extend the mapping of the Myc interactome and contribute to a greater understanding of Myc regulation by protein-protein interactions. Furthermore, we are currently in the process of employing this technique to map the interactomes of two other Myc family members, N-Myc and L-Myc. We are interested in identifying common interactors of the Myc family members that contribute to their oncogenic activity, validate them, and explore whether these interactors could be potential therapeutic targets in cancers with deregulated Myc activity. Citation Format: Diana Resetca, Dharmesh Dingar, Manpreet Kalkat, Brian Raught, Linda Z. Penn. Charactering the interactomes of the Myc family of oncogenes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2010.</jats:p

Abstract A12: Identifying MYC post-translational modifications using a mass spectrometry-based approach

Abstract MYC activity is regulated by a complex network of signaling cascades and protein interactions, some of which result in post-translation modifications (PTMs). For example, phosphorylation of the regulatory threonine 58 (T58) and serine 62 (S62) plays a pivotal role in regulating MYC stability and activity. Loss of this regulatory pathway in cancer can lead to MYC dysregulation and contribute to tumorigenesis. Despite their important role, the majority of MYC PTMs arising downstream of different signaling cascades and their consequences on regulating MYC activity remain largely unknown. To capture the broad spectrum of MYC PTMs, a mass spectrometry (MS)-based approach is being pursued to attain high sequence coverage and enable the identification of PTMs throughout the protein. MYC was overexpressed in the HEK293T cell line, immunoprecipitated, digested, and analyzed on the Velos Orbitrap MS. This analysis facilitated the identification of a range of MYC PTMs, including phosphorylation, sumoylation, ubiquitination and acetylation. Phosphorylation was readily detectable at residues T58 and S62, consistent with previous reports. Additionally, we also observed a previously reported phosphorylation cluster involving residues T343/S344/S347/S348, suggesting that these sites might play a role in regulating MYC function. Indeed, converting these residues to alanine to prevent phosphorylation resulted in a gain-of-function mutant (See Penn Lab poster Lorenco et al.). Another phosphorylation was observed mapping to residues S71 and/or S81. Mutating these sites to alanine also potentiated MYC transformation, highlighting the role of PTMs in regulating MYC function. A MYC sumoylation site was identified on lysine 326 (K326) and is subject of ongoing investigation (See Penn Lab poster Kalkat et al.). Additionally, we will discuss approaches presently underway to increase sequence coverage and identify additional MYC PTMs. PTMs play an important role in regulating the activity of transcription factors, including that of MYC. Moreover, PTMs can contribute to the dysregulation of its activity in cancer. Thus, mapping the array of PTMs in MYC, understanding their role in regulating MYC function, and elucidating the pathways that converge on these PTMs may pave the way for the development of novel therapeutic strategies aimed at targeting MYC-induced tumorigenesis. Citation Format: Diana Resetca, Manpreet Kalkat, Corey Lourenco, Pak-Kei Chan, Tharan Srikumar, Brian Raught, Linda Penn. Identifying MYC post-translational modifications using a mass spectrometry-based approach. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A12.</jats:p