Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments

Ribosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.

A novel translation inhibitor, mersicarpine, inhibits S-phase progression and induces apoptosis in HL60 cells

Mersicarpine is an aspidosperma alkaloid isolated from the Kopsia genus of plants. Its intriguing structural features have attracted much attention in synthetic organic chemistry, but no biological activity has been reported. Here, we report the effects of mersicarpine on human leukemia cell line HL60. At concentrations above 30 µm, mersicarpine reversibly arrested cell cycle progression in S-phase. At higher concentrations, it induced not only production of reactive oxygen species, but also apoptosis. Macromolecular synthesis assay revealed that mersicarpine specifically inhibits protein synthesis. These results suggest that mersicarpine is a novel translation inhibitor that induces apoptosis.

Puromycin reactivity does not accurately localize translation at the subcellular level

Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.

Puromycin reactivity does not accurately localize translation at the subcellular level

Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Here, using in vitro and in vivo experiments, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in the in vivo context.

Broad Efficacy of the Translation Inhibitor Omacetaxine in Relapsed/Refractory Multiple Myeloma Samples

Introduction: The prognosis of patients with multiple myeloma (MM) who are resistant to proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs) is extremely poor. Even daratumumab and BCMA CAR-T cell therapies appear to provide only temporary benefit before patients eventually succumb to their disease. We show that protein translation is upregulated in MM cells. We also demonstrate that translational inhibition with omacetaxine (Oma) is specifically cytotoxic in a manner that is preserved in MM cells from PI/IMiD resistant patients. Furthermore, protein translation inhibition is synergistic with IMiDs, even in IMiD refractory patients. We hypothesize these findings stem from the origin of MM cells as professional antibody secretory cells, with highly active protein translation fundamental to their biology. Here, we show these findings and provide support to test the translation inhibitor omacetaxine with a clinical trial in multiple myeloma patients. Methods: Primary MM cells in mononuclear cell (MNC) cultures were assayed ex vivo for Oma effect at 48-hrs by high-throughput flow cytometry using our myeloma drug sensitivity testing (My-DST) platform. Protein synthesis rates were measured via flow cytometry using the Cayman OP-puromycin assay. Initial drug combination matrices were performed in MM cell lines via MTS assay after 96-hr incubation. Cell line xenograft study of the Oma and IMiD combination in vivo were performed using an orthometastatic MM model with MM1.S luciferase-expressing cells injected IV in NSG mice. Vehicle control, Oma, pomalidomide (Pom) and the combination were dosed 5 days/week by IP injection beginning on day 30. Disease burden was monitored with imaging of luciferase bioluminescence. Results: Oma had specific cytotoxicity across 5 MM cell lines (EC50 = 15-35 nM, data not shown). Oma also showed MM cell specific cytotoxicity in patient samples with EC50 ranging from 25-100 nM (Fig 1A). Ex vivo treatment with 50 nM Oma for 48 hrs caused >50% reduction in viable MM cells in 39/50 (78%) patient samples. Importantly, 50 nM Oma retained activity in PI/IMiD-refractory patients compared to sensitive patients (64.2% vs 71.6% mean decrease in MM cell viability, respectively, P = 0.44, Fig 1B). Primary MM cells exhibited significantly higher levels of baseline protein translation than other bone marrow MNCs with mean 3.9-fold increase (n = 14, P = <0.001) (Fig 1C). Oma treatment inhibited protein translation in a dose dependent manner after 2.5 hrs in H929 cells with EC50 9 nM (Fig 1D), and translation in the primary MM cells was significantly reduced after 2.5 hrs of 50 nM Oma treatment to near MNC levels (Fig 1E). Using a cutoff of 2.5-fold higher baseline MM translation compared to MNCs, "High Translation" was associated with sensitivity to Oma, and "Low Translation" was associated with relative resistance (P = 0.0018) (Fig 1F). Provocatively, Oma demonstrated synergy in combination with Pom (5.52 ZIP synergy δ-score) in the MM1.S cell line (Fig 1G), but was antagonistic with PIs (data not shown). Notably, in an IMiD-resistant relapsed patient sample, the combination of Oma and Pom was even more synergistic and re-sensitized the MM cells to the IMiD (22.68 δ-score, Fig 1H). In a MM1.S luciferase xenograft model of advanced MM, Oma treatment significantly extended survival compared to vehicle (HR = 6.67, P = 0.021), Pom treatment led to a trend towards extended survival (HR = 2.78, P = 0.16), and Oma in combination with Pom extended median survival the most (HR = 16.98, P = 0.002) (Fig 1I-J). Conclusion: Protein translation inhibitors represent a potential new drug class for clinical myeloma treatment. We have shown that the protein synthesis inhibitor omacetaxine possesses a potent and specific killing effect against MM cells. Importantly, this anti-myeloma cytotoxicity was equally active in biopsy samples obtained from patients at diagnosis and patients with disease advanced to the late stage of PI/IMiD resistance. The pretreatment level of MM cell protein translation may serve as a biomarker for clinical response. In further exploration of the optimized treatment approach for clinical trials, we found synergy between omacetaxine and pomalidomide in vitro, in vivo and using patient samples ex vivo. A phase I clinical trial of omacetaxine single agent and in combination with pomalidomide is currently in development at the University of Colorado. Disclosures Mark: Janssen: Honoraria; Takeda: Honoraria; Amgen: Honoraria; Celgene: Honoraria.

Epigenetic regulation of protein translation in KMT2A-rearranged AML

ABSTRACTInhibition of the histone methyl-transferase DOT1L (KMT4) has shown encouraging activity in preclinical models of KMT2A (MLL)-rearranged leukemia. The DOT1L inhibitor pinometostat (EPZ5676) was well tolerated in early phase clinical trials and showed modest clinical activity, including occasional complete responses (CRs) as single agent. These studies support the development of combinatorial therapies for KMT2A-rearranged leukemias. Here, we investigated two novel combinations: dual inhibition of the histone methyltransferases DOT1L and EZH2, and the combination of a DOT1L inhibitor with the protein synthesis inhibitor homoharringtonine (HHR).EZH2 is the catalytic histone methyltransferase in the polycomb repressive complex 2 (PRC2), and inhibition of EZH2 has reported preclinical activity in KMT2A-rearranged leukemia. We found that the H3K79 and H3K27 methyl marks are not dependent on each other, and that DOT1L and EZH2 inhibition affect largely distinct gene expression programs. In particular, the KMT2A/DOT1L target HOXA9, which is commonly de-repressed as a consequence of PRC2 loss or inhibition in other contexts, was not re-activated upon dual DOT1L/EZH2 knockout or inhibition. Despite encouraging data in murine KMT2A-MLLT3 transformed cells suggesting synergy between DOT1L and EZH2 inhibition, we found both synergistic and antagonistic effects on a panel of human KMT2A rearranged cell lines. Combinatorial inhibition of DOT1L and EZH2 is thus not a promising strategy. We identified opposing effects on ribosomal gene transcription and protein translation by DOT1L and EZH2 as a mechanism that is partially responsible for observed antagonistic effects. The effects of DOT1L inhibition on ribosomal gene expression prompted us to evaluate the combination of EPZ5676 with a protein translation inhibitor. EPZ5676 was synergistic with the protein translation inhibitor homoharringtonine (HHR), supporting further preclinical/clinical development of this combination.

The translation inhibitor cycloheximide affects ribosome profiling data in a species-specific manner

Ribosome profiling provides genome-wide snapshots of translation dynamics by determining ribosomal positions at sub-codon resolution. To maintain this positional information, the translation inhibitor cycloheximide (CHX) has been widely used to arrest translating ribosomes prior to library preparation. Several studies have reported CHX-induced biases in yeast data casting uncertainty about its continued use and questioning the accuracy of many ribosome profiling studies. However, the presence of these biases has not been investigated comprehensively in organisms other than Saccharomyces cerevisiae. Here, we use a highly standardized and optimized protocol to compare different CHX-treatment conditions in yeast and human cells. Our data suggest that unlike in S. cerevisiae, translating ribosomes in human cells are not susceptible to conformational restrictions by CHX. Furthermore, CHX-induced codon-specific effects on ribosome occupancy are not detectable in human cells nor in other model organisms including Schizosaccharomyces pombe and Candida albicans. In fact, we find that even in S. cerevisiae most biases can be avoided by omitting CHX pre-treatment, indicating that other parameters of library generation contribute to differences between ribosome profiling experiments. Together our findings provide a framework for researchers who plan their own ribosome profiling experiments or who analyze published datasets to draw judicious conclusions.