TP53 is commonly mutated in cancer, giving rise to loss of wild-type tumor suppressor function and increases in gain-of-function oncogenic roles. Thus, inhibition of mutant p53 and reactivation of wild-type function represents a potential means to target diverse tumor types. (E)-1-(4-Methylpiperazin-1-yl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one (NSC59984), first identified from a high-throughput screen, induces wild-type p53 signaling and antiproliferative effects while inhibiting mutant p53 gain-of-function activities. Here, we investigate the specific mechanism of action of NSC59984 against p53. We found that NSC59984 reacts with thiols via an unusual Michael addition at the alpha-carbon. Covalent modification of p53 Cys124 and Cys229 was observed both following in vitro reaction and upon treatment of cells. Finally, we used a biotinylated form of NSC59984 and, separately, thermal proteome profiling to examine off-target effects, identifying several metabolic proteins involved in cellular metabolism as potential targets. These results demonstrate that covalent modification of p53 by NSC59984 leads to increased wild-type activity and suggest that potential reaction with metabolic enzymes may contribute to antiproliferative function.

Supplemental tables 1-3, supplemental figure legends, supplemental figures 1-4

TP53 is the most commonly mutated gene in cancer, and gain-of-function mutations have wide-ranging effects. Efforts to reactivate wild-type p53 function and inhibit mutant functions have been complicated by the variety of TP53 mutations. Identified from a screen, the NSC59984 compound has been shown to restore activity to mutant p53 in colorectal cancer cells. Here, we investigated its effects on esophageal adenocarcinoma cells with specific p53 hot-spot mutations. NSC59984 treatment of cells reactivated p53 transcriptional regulation, inducing mitochondrial intrinsic apoptosis. Analysis of its effects on cellular metabolism demonstrated increased utilization of the pentose phosphate pathway and inhibition of glycolysis at the fructose-1,6-bisphosphate to fructose 6-phosphate junction. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; these effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. We found that the effects of NSC59984 were substantially greater in cells harboring the p53 R248W mutation. Overall, these findings demonstrate p53-dependent effects of NSC59984 on cellular metabolism, with increased activity in cells harboring the p53 R248W mutation. This research highlights the importance of defining the mutational status of a particular cancer to create a patient-centric strategy for the treatment of p53-driven cancers.

The tumor suppressor p53 is mutated (mt-p53) in over 50% of human cancers causing gain-of-function oncogenic effects, including metabolic changes that reduce tumor responsiveness to radio/chemotherapy. Common hot-spot mutations within the DNA-binding domain can be categorized as conformational (R175H) or DNA binding (R248W). NSC59984 has been characterized as a small molecule that targets mt-p53 for degradation and restores wt-p53 signaling. Using esophageal adenocarcinoma cells and CRISPR generated isogenic cell lines bearing matching hot-spot p53 mutations, we aim to understand how the molecular features of mt-p53 affect drug efficiency and enable the development of targeted therapies to limit cancer cell growth. We found that NSC59984 covalently modifies p53 by Michael addition at cysteine residues 124 and 229, which promote interactions that would stabilize the protein/DNA complex leading to increased p53 transcriptional activity. In cells, the effects of NSC59984 were substantially greater in cells harboring the R248W mutation compared with the R175H mutation. Treatment with NSC59984 reduced proliferation and increased apoptosis via the intrinsic mitochondrial pathway. It also induced changes in OXPHOS, ATP level, mitochondrial membrane potential, glycolysis, and lactate production. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. NSC59984 treatment increased G6PD activity, total NADPH levels, and expression of TIGAR. Knockout of TIGAR partially removed the antiproliferative effects of the drug and reduced G6PD levels in the p53-R248W cells. Incorporation of [13C6] into cellular metabolites suggests that p53-regulated transcription of TIGAR increased utilization of the pentose phosphate pathway and inhibited glycolysis at the fructose-6-P fructose-1,6-bisphosphate junction, supported by an increase in Hexokinase 2 and a decrease of phosphofructokinase-1. Thermal proteome profiling identified TIGAR as an additional reaction target of NSC59984, suggesting increased involvement in modulating these metabolic effects. Combining currently available therapeutic metabolic inhibitors with NSC59984 enhanced the antiproliferative effects in cells harboring p53-R248W creating a therapeutic window when compared to the wt-p53 expressing cells. This suggests these combinations could be used in a clinically relevant setting. Overall, this work has identified a distinctive mode of action for p53 reactivation resulting in not only transcriptional activity, but also a unique effect on cellular energetics. This study shows evidence of variation in responsiveness of different mt-p53 forms and allows the development of specific therapeutics directed to individuals for patient-centered precision medicine. Importantly, we have shown that targeting p53 signaling has significant effects on the metabolic profiles of cancer cells rendering them more vulnerable to neoadjuvant therapy. Citation Format: Kate Brown, Lisa Jenkins, Dan Crooks, Deborah Surman, Sharlyn Mazur, Yuan Xu, Bhargav Arimilli, Ye Yang, Andrew Lane, Stewart Durell, Teresa Fan, David Schrump, Marston Marston, Taylor Ripley, Ettore Appella, Gaelyn Lyons, Andrew Perciaccante, Jerry Dinan, Marco Robello, Herman Nikolayevskiy, Robert O’Connor, Daniel Appella. Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A103.

T cell precursors develop into helper CD4 or cytotoxic CD8 T cells in thymus. Thpok, a BTB/POZ family transcription factor, enforces MHC II restricted thymocytes commitment to the CD4 lineage, while Runx3 protein drives MHC I restricted thymocytes to the CD8 lineage. However, the molecular mechanisms underlying Thpok functions during CD4 T cell development are not understood. Replacing the BTB domain from Thpok with that from Bcl6, another BTB/POZ zinc finger transcription factor that recruits NCOR-family co-repressors through its BTB domain, abrogated CD4 T cell development in thymus; this suggests a unique feature associated with Thpok BTB domain. Here, using mass-spectrometry, we identified nucleosome remodeling and deacetylase (NuRD) complex as a novel Thpok cofactor. We demonstrated that the Thpok BTB domain is essential for NuRD recruitment. Moreover, we identified BTB domain amino acid residues critical for NuRD complex interaction, and showed that mutation of these Thpok residues disrupts CD4 T cell development in vivo. Reciprocally, a fusion protein reconstituting NuRD binding to a BTB-less version of Thpok (which cannot bind NuRD) restored CD4 T cells in vivo. In addition, we found that CD4-lineage thymocytes expressing that fusion protein have a transcriptome similar to that of thymocytes expressing wild type Thpok. Finally, we found that NuRD binding is required for Thpok to antagonize Runx3 expression in thymocytes. Thus, our results demonstrate that NuRD recruitment is both necessary and sufficient for the functions of the Thpok BTB domain in CD4 T cell development and it is also required to antagonize Runx3-dependent differentiation towards CD8 lineage.

Abstract Relapsed pediatric rhabdomyosarcomas (RMS) and neuroblastomas (NBs) have a poor prognosis despite multimodality therapy. In addition, the current standard of care for these cancers includes vinca alkaloids that have severe toxicity profiles, further underscoring the need for novel therapies for these malignancies. Here, we show that the small-molecule rigosertib inhibits the growth of RMS and NB cell lines by arresting cells in mitosis, which leads to cell death. Our data indicate that rigosertib, like the vinca alkaloids, exerts its effects mainly by interfering with mitotic spindle assembly. Although rigosertib has the ability to inhibit oncogenic RAS signaling, we provide evidence that rigosertib does not induce cell death through inhibition of the RAS pathway in RAS-mutated RMS and NB cells. However, the combination of rigosertib and the MEK inhibitor trametinib, which has efficacy in RAS-mutated tumors, synergistically inhibits the growth of an RMS cell line, suggesting a new avenue for combination therapy. Importantly, rigosertib treatment delays tumor growth and prolongs survival in a xenograft model of RMS. In conclusion, rigosertib, through its impact on the mitotic spindle, represents a potential therapeutic for RMS.

Natural killer (NK) cells play key roles in immune surveillance against tumors and viral infection. NK cells distinguish abnormal cells from healthy cells by cell–cell interaction with cell surface proteins and then attack target cells via multiple mechanisms. In addition, extracellular vesicles (EVs) derived from NK cells (NK-EVs), including exosomes, possess cytotoxic capacity against tumor cells, but their characteristics and regulation by cytokines remain unknown. Here, we report that EVs derived from human NK-92 cells stimulated with IL-15 + IL-21 show enhanced cytotoxic capacity against tumor cells. Major cytolytic granules, granzyme B and granzyme H, are enriched by IL-15 + IL-21 stimulation in NK-EVs; however, knockout experiments reveal those cytolytic granules are independent of enhanced cytotoxic capacity. To find out the key molecules, mass spectrometry analyses were performed with different cytokine conditions, no cytokine, IL-15, IL-21, or IL-15 + IL-21. We then found that CD226 (DNAM-1) on NK-EVs is enriched by IL-15 + IL-21 stimulation and that blocking antibodies against CD226 reduced the cytolytic activity of NK-EVs. We also show NK-EVs are taken up by target cells via macropinocytosis. Collectively, our findings elucidate the novel properties of NK-EVs and the mechanism of their incorporation into target cells.

Additional file 7: Table S3. List of all identified proteins pulled down with TRAP1 using an IP-MS analysis with WT TRAP1, and the TRAP1 mutants E115A/R402A and ΔStrap.

Background The molecular chaperone TRAP1, the mitochondrial isoform of cytosolic HSP90, remains poorly understood with respect to its pivotal role in the regulation of mitochondrial metabolism. Most studies have found it to be an inhibitor of mitochondrial oxidative phosphorylation (OXPHOS) and an inducer of the Warburg phenotype of cancer cells. However, others have reported the opposite, and there is no consensus on the relevant TRAP1 interactors. This calls for a more comprehensive analysis of the TRAP1 interactome and of how TRAP1 and mitochondrial metabolism mutually affect each other. Results We show that the disruption of the gene for TRAP1 in a panel of cell lines dysregulates OXPHOS by a metabolic rewiring that induces the anaplerotic utilization of glutamine metabolism to replenish TCA cycle intermediates. Restoration of wild-type levels of OXPHOS requires full-length TRAP1. Whereas the TRAP1 ATPase activity is dispensable for this function, it modulates the interactions of TRAP1 with various mitochondrial proteins. Quantitatively by far, the major interactors of TRAP1 are the mitochondrial chaperones mtHSP70 and HSP60. However, we find that the most stable stoichiometric TRAP1 complex is a TRAP1 tetramer, whose levels change in response to both a decline and an increase in OXPHOS. Conclusions Our work provides a roadmap for further investigations of how TRAP1 and its interactors such as the ATP synthase regulate cellular energy metabolism. Our results highlight that TRAP1 function in metabolism and cancer cannot be understood without a focus on TRAP1 tetramers as potentially the most relevant functional entity.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely utilized technique for macromolecule and protein analysis. While multiple methods exist to visualize the separated protein bands on gels, one of most popular methods of staining the proteins is with Coomassie dye. A more recent approach is to use Bio-Rad stain-free technology for visualizing protein bands with UV light and achieve similar or greater sensitivity than that of Coomassie dye. Here, we developed a method to further amplify the sensitivity of stain-free gels using carboxyfluorescein succinimidyl ester (CFSE) staining. We compared our novel method using foetal bovine serum samples with Coomassie dye, standard stain-free gels, and silver staining. Our results show that while silver staining remains a gold-standard method in terms of sensitivity; CFSE staining of samples prior to use with stain-free gels results in a 10–100-fold increase in sensitivity over Coomassie staining and the standard stain-free method. Our method offers a sensitivity similar to that of silver staining which is compatible with downstream mass spectrometry, and therefore more advantageous for further retrieval and analysis of macromolecules in bands.

Additional file 13: Table S8. TRAP1 complex MS analysis.

Cells respond to ionizing radiation (IR) through the activities of constitutively expressed proteins and through changes in gene expression. Constitutively expressed proteins play well-defined roles in major components of the cellular radioresponse, such as DNA repair and cell cycle. However, the contribution of changes in gene expression to the radioresponse is less understood. We have previously demonstrated through polysome profiling and microarray analysis that translational control is a key component of radiation-induced changes in gene expression. In response to IR, specific transcripts are recruited to or away from polysomes without affecting the global polysome profile of the cells. One mechanistic explanation for this observation is that IR influences the activity of RNA binding proteins (RBPs), which regulate the inclusion or exclusion of transcripts from polysomes. Here, we utilized RNA interactome capture (RIC) to identify RBPs bound to transcripts in control and irradiated human cancer cells. Cells were either mock irradiated or treated with 2Gy. One hour post-IR, RNA and protein complexes were crosslinked with 254 nm UV light. mRNA was isolated from cell lysates with oligo(dT) magnetic beads followed by several rounds of highly stringent washes to remove non-specific interactors. RNA was removed from crosslinked proteins by RNase digestion. The proteins were separated on SDS-PAGE gels and subjected to in-gel trypsin digestion, and the subsequent peptides were analyzed by label-free quantification mass spectrometry using an Orbitrap Fusion mass spectrometer. Proteome Discoverer 2.2 was used to search the data against human proteins from the UniProt database using SequestHT with a 1% false discovery rate. We found that we were able to efficiently pull down RNA:protein complexes with a crosslinking dose of 150 mJ/cm2. As determined by silver staining, the protein pattern of crosslinked samples differed greatly from protein isolated from whole cell lysates. By western blot, this method highly enriched for known RBPs, such as PTBP1 and CUGBP1, while contamination from non-RBPs, such as actin and tubulin, was not detected. By mass spectrometry, we identified hundreds of RBPs in each replicate above the no crosslinking background control for both untreated and irradiated cells. However, more RBPs were consistently detected in the control cells. Examples of RBPs preferentially binding in control cells include SSB, a protein involved in a variety of RNA metabolic processes, and U2AF2, a protein involved in RNA splicing. Overall, these data demonstrate that RIC is a sensitive and rigorous method to identify RBPs and that RBPs may play a role in radiation-induced post-transcriptional gene regulation. Preferential binders identified by this method may provide further insight into pathways regulating tumor cell radiosensitivity. Citation Format: Stacey L. Lehman, Theresa Wechsler, Gaelyn C. Lyons, Lisa M. Jenkins, Kevin Camphausen, Philip J. Tofilon. Identification of RNA binding proteins influenced by ionizing radiation through RNA interactome capture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3741.

For HIV to become infectious, any new virion produced from an infected cell must undergo a maturation process that involves the assembly of viral polyproteins Gag and Gag-Pol at the membrane surface. The self-assembly of these viral proteins drives formation of a new viral particle as well as the activation of HIV protease, which is needed to cleave the polyproteins so that the final core structure of the virus will properly form. Molecules that interfere with HIV maturation will prevent any new virions from infecting additional cells. In this manuscript, we characterize the unique mechanism by which a mercaptobenzamide thioester small molecule (SAMT-247) interferes with HIV maturation via a series of selective acetylations at highly conserved cysteine and lysine residues in Gag and Gag-Pol polyproteins. The results provide the first insights into how acetylation can be utilized to perturb the process of HIV maturation and reveal a new strategy to limit the infectivity of HIV.