Lenalidomide is a drug with clinical efficacy in multiple myeloma and other B cell neoplasms, but its mechanism of action is unknown. Using quantitative proteomics, we found that lenalidomide causes selective ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IKZF3, by the CRBN-CRL4 ubiquitin ligase. IKZF1 and IKZF3 are essential transcription factors in multiple myeloma. A single amino acid substitution of IKZF3 conferred resistance to lenalidomide-induced degradation and rescued lenalidomide-induced inhibition of cell growth. Similarly, we found that lenalidomide-induced IL2 production in T cells is due to depletion of IKZF1 and IKZF3. These findings reveal a novel mechanism of action for a therapeutic agent, alteration of the activity of an E3 ubiquitin ligase leading to selective degradation of specific targets.

The “cancerized field” concept posits that cells in a given tissue share an oncogenic mutation or insult and are thus cancer-prone, yet only discreet clones within the field initiate tumors. Nearly all benign nevi carry oncogenic BRAFV600E mutations, but they only rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCP’s) and is specifically re-expressed in melanoma. We show by live imaging of transgenic zebrafish crestin reporters that, within a cancerized field (BRAFV600E-mutant; p53-deficient), a single melanocyte reactivates the NCP state, and this establishes that a fate change occurs at melanoma initiation in this model. We show the crestin element is regulated by NCP transcription factors, including sox10. Forced sox10 overexpression in melanocytes accelerated melanoma formation, consistent with activation of a NCP gene signature and super-enhancers leading to melanoma. Our work highlights the importance of NCP state reemergence as a key event in melanoma initiation.

SUMMARY Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3β inhibitors, two calpain inhibitors and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin and the GSK3β inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle.

The design of modular protein logic for regulating protein function at the posttranscriptional level is a challenge for synthetic biology. Here, we describe the design of two-input AND, OR, NAND, NOR, XNOR, and NOT gates built from de novo–designed proteins. These gates regulate the association of arbitrary protein units ranging from split enzymes to transcriptional machinery in vitro, in yeast and in primary human T cells, where they control the expression of the TIM3 gene related to T cell exhaustion. Designed binding interaction cooperativity, confirmed by native mass spectrometry, makes the gates largely insensitive to stoichiometric imbalances in the inputs, and the modularity of the approach enables ready extension to three-input OR, AND, and disjunctive normal form gates. The modularity and cooperativity of the control elements, coupled with the ability to de novo design an essentially unlimited number of protein components, should enable the design of sophisticated posttranslational control logic over a wide range of biological functions.

SUMMARY Apart from their antimicrobial properties, tetracyclines demonstrate clinically validated effects in the amelioration of pathological inflammation and human cancer. Delineation of the target(s) and mechanism(s) responsible for these effects, however, has remained elusive. Here, employing quantitative mass spectrometry-based proteomics, we identified human 80S ribosomes as targets of the tetracyclines Col-3 and doxycycline. We then developed in-cell click selective crosslinking with RNA sequence profiling (icCL-Seq) to map binding sites for these tetracyclines on key human rRNA substructures at nucleotide resolution. Importantly, we found that structurally and phenotypically variant tetracycline analogs could chemically discriminate these rRNA binding sites. We also found that tetracyclines both subtly modify human ribosomal translation and selectively activate the cellular integrated stress response (ISR). Together, the data reveal that targeting of specific rRNA substructures, activation of the ISR, and inhibition of translation are correlated with the anti-proliferative properties of tetracyclines in human cancer cell lines.

Phenotypic cell-based screening is a powerful approach to small-molecule discovery, but a major challenge of this strategy lies in determining the intracellular target and mechanism of action (MoA) for validated hits. Here, we show that the small-molecule BRD0476, a novel suppressor of pancreatic β-cell apoptosis, inhibits interferon-gamma (IFN-γ)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote β-cell survival. However, unlike common JAK-STAT pathway inhibitors, BRD0476 inhibits JAK-STAT signaling without suppressing the kinase activity of any JAK. Rather, we identified the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) as an intracellular target, using a quantitative proteomic analysis in rat β cells. RNAi-mediated and CRISPR/Cas9 knockdown mimicked the effects of BRD0476, and reverse chemical genetics using a known inhibitor of USP9X blocked JAK-STAT signaling without suppressing JAK activity. Site-directed mutagenesis of a putative ubiquitination site on JAK2 mitigated BRD0476 activity, suggesting a competition between phosphorylation and ubiquitination to explain small-molecule MoA. These results demonstrate that phenotypic screening, followed by comprehensive MoA efforts, can provide novel mechanistic insights into ostensibly well-understood cell signaling pathways. Furthermore, these results uncover USP9X as a potential target for regulating JAK2 activity in cellular inflammation.

The neural crest is a dynamic progenitor cell population that arises at the border of neural and non-neural ectoderm. The inductive roles of FGF, Wnt, and BMP at the neural plate border are well established, but the signals required for subsequent neural crest development remain poorly characterized. Here, we conducted a screen in primary zebrafish embryo cultures for chemicals that disrupt neural crest development, as read out by crestin:EGFP expression. We found that the natural product caffeic acid phenethyl ester (CAPE) disrupts neural crest gene expression, migration, and melanocytic differentiation by reducing Sox10 activity. CAPE inhibits FGF-stimulated PI3K/Akt signaling, and neural crest defects in CAPE-treated embryos are suppressed by constitutively active Akt1. Inhibition of Akt activity by constitutively active PTEN similarly decreases crestin expression and Sox10 activity. Our study has identified Akt as a novel intracellular pathway required for neural crest differentiation.

Lenalidomide is a highly effective drug for the treatment of multiple myeloma and has activity in additional B cell lymphomas. Lenalidomide has been shown to bind the CRBN-DDB1 E3 ubiquitin ligase, but it is unknown how lenalidomide alters the activity of this enzyme complex, and how this leads to therapeutic efficacy.  We used a combination of quantitative proteomic approaches to demonstrate that lenalidomide acts by a novel mechanism of action for a therapeutic agent: in multiple myeloma cells, lenalidomide increases the binding of two substrates, IKZF1 (Ikaros) and IKZF3 (Aiolos), to the CRBN substrate adaptor; increases the ubiquitination of these substrates; and causes the targeted degradation of these transcription factors that are essential for the differentiation and survival of plasma cells including multiple myeloma cells. To identify targets of the CRBN-DDB1 ubiquitin ligase that are altered by lenalidomide, we applied SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative mass spectrometry studies to globally assess changes in ubiquitination and proteome levels in the multiple myeloma cell line MM1S. Two members of the Ikaros transcription factor family, IKZF1 and IKZF3, were differentially ubiquitinated and decreased after lenalidomide treatment.  Subsequent validation experiments in various cell lines demonstrated that lenalidomide, thalidomide, and pomalidomide cause a decrease of endogenous and ectopically expressed IKZF1 and IKZF3 protein levels but not mRNA levels. Furthermore, we confirmed that IKZF1 and IKZF3 bind CRBN in the presence of lenalidomide, supporting CRBN’s role as a substrate adaptor. Consistent with this, shRNA mediated knockdown or overexpression of a CRBN mutant (CRBNYWAA) that does not bind lenalidomide abrogated lenalidomide’s effect on IKZF1 and IKZF3. Moreover, CRBN promoted IKZF3 ubiquitination in vitro in the presence of lenalidomide, demonstrating that it is an enzymatic substrate. Using deletion mutants of IKZF3 we identified a 58-amino-acid degron in the N-terminal zinc finger domain that is sufficient for lenalidomide-induced degradation. Based on sequence alignment of that region between lenalidomide responding Ikaros proteins IKZF1 and IKZF3 vs. non-responding IKZF2, IKZF4 and IKZF5 we substituted a single amino acid (IKZF3Q147H) that prevented binding of IKZF3 to CRBN and conferred resistance to lenalidomide induced degradation. IKZF1 and IKZF3 are essential transcription factors for terminal B cell differentiation. We evaluated the biological effects of IKZF1 and IKZF3 loss using shRNAs in a variety of cell lines. IKZF1 and IKZF3 specific shRNAs inhibited the growth of multiple myeloma cell lines while lenalidomide insensitive cell lines derived from other hematopoietic neoplasms were unaffected. Similarly, a dominant negative IKZF3 mutant resulted in growth inhibition of MM1S cells. In contrast, expression of IKZF3Q147Hconferred lenalidomide resistance to MM1S cells. Lenalidomide induces IL-2 expression and release in T cells. We found that lenalidomide induced a dose-dependent decrease of IKZF1 and IKZF3 protein levels in primary human T cells. Previous studies have shown that IKZF3 is a transcriptional repressor of IL-2. To further evaluate the effect of IKZF3 loss, we transduced primary human T cells with shRNAs targeting either IKZF3 or control. IL2 RNA levels increased 3.3 fold after lenalidomide treatment in T cells expressing control shRNAs. In contrast, the baseline IL2 RNA level in T cells transduced with IKZF3 specific shRNAs was 3.7 fold higher compared to controls and this effect could not be further stimulated by lenalidomide. In conclusion, selective targeting of two lymphoid transcription factors, IKZF1 and IKZF3, explains lenalidomide’s selective growth inhibition in multiple myeloma and likely other B cell lymphomas as well as its immunomodulatory effects in T cells. Furthermore, selective ubiquitination and degradation of specific targets provides a novel mechanism of therapeutic activity for proteins that are not otherwise amenable to small-molecule inhibition. Disclosures: Ebert: Celgene: Membership on an entity’s Board of Directors or advisory committees.