Fei Ji

Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition

Although mechanisms of acquired resistance of epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. Here we observe that acquired resistance caused by the EGFRT790M gatekeeper mutation can occur either by selection of pre-existing EGFRT790M-positive clones or via genetic evolution of initially EGFRT790M-negative drug-tolerant cells. The path to resistance impacts the biology of the resistant clone, as those that evolved from drug-tolerant cells had a diminished apoptotic response to third-generation EGFR inhibitors that target EGFRT790M; treatment with navitoclax, an inhibitor of the anti-apoptotic factors BCL-xL and BCL-2 restored sensitivity. We corroborated these findings using cultures derived directly from EGFR inhibitor–resistant patient tumors. These findings provide evidence that clinically relevant drug-resistant cancer cells can both pre-exist and evolve from drug-tolerant cells, and they point to therapeutic opportunities to prevent or overcome resistance in the clinic.

Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation

Master regulatory genes require stable silencing by the polycomb group (PcG) to prevent misexpression during differentiation and development. Some PcG proteins covalently modify histones, which contributes to heritable repression. The role for other effects on chromatin structure is less understood. We characterized the organization of PcG target genes in ESCs and neural progenitors using 5C and super-resolution microscopy. The genomic loci of repressed PcG targets formed discrete, small (20-140 Kb) domains of tight interaction that corresponded to locations bound by canonical polycomb repressive complex 1 (PRC1). These domains changed during differentiation as PRC1 binding changed. Their formation depended upon the Polyhomeotic component of canonical PRC1 and occurred independently of PRC1-catalyzed ubiquitylation. PRC1 domains differ from topologically associating domains in size and boundary characteristics. These domains have the potential to play a key role in transmitting epigenetic silencing of PcG targets by linking PRC1 to formation of a repressive higher-order structure.

IGF2 mRNA binding protein-2 is a tumor promoter that drives cancer proliferation through its client mRNAs IGF2 and HMGA1

The gene encoding the Insulin-like Growth Factor 2 mRNA binding protein 2/IMP2 is amplified and overexpressed in many human cancers, accompanied by a poorer prognosis. Mice lacking IMP2 exhibit a longer lifespan and a reduced tumor burden at old age. Herein we show in a diverse array of human cancer cells that IMP2 overexpression stimulates and IMP2 elimination diminishes proliferation by 50–80%. In addition to its known ability to promote the abundance of Insulin-like Growth Factor 2/IGF2, we find that IMP2 strongly promotes IGF action, by binding and stabilizing the mRNA encoding the DNA binding protein HMGA1, a known oncogene. HMGA1 suppresses the abundance of IGF binding protein 2/IGFBP2 and Grb14, inhibitors of IGF action. IMP2 stabilization of HMGA1 mRNA plus IMP2 stimulated IGF2 production synergistically drive cancer cell proliferation and account for IMP2’s tumor promoting action. IMP2’s ability to promote proliferation and IGF action requires IMP2 phosphorylation by mTOR.

PhyloGene server for identification and visualization of co-evolving proteins using normalized phylogenetic profiles

Proteins that function in the same pathways, protein complexes or the same environmental conditions can show similar patterns of sequence conservation across phylogenetic clades. In species that no longer require a specific protein complex or pathway, these proteins, as a group, tend to be lost or diverge. Analysis of the similarity in patterns of sequence conservation across a large set of eukaryotes can predict functional associations between different proteins, identify new pathway members and reveal the function of previously uncharacterized proteins. We used normalized phylogenetic profiling to predict protein function and identify new pathway members and disease genes. The phylogenetic profiles of tens of thousands conserved proteins in the human, mouse, Caenorhabditis elegans and Drosophila genomes can be queried on our new web server, PhyloGene. PhyloGene provides intuitive and user-friendly platform to query the patterns of conservation across 86 animal, fungal, plant and protist genomes. A protein query can be submitted either by selecting the name from whole-genome protein sets of the intensively studied species or by entering a protein sequence. The graphic output shows the profile of sequence conservation for the query and the most similar phylogenetic profiles for the proteins in the genome of choice. The user can also download this output in numerical form.

Maintenance of macrophage transcriptional programs and intestinal homeostasis by epigenetic reader SP140

The epigenetic reader SP140, which is polymorphic in Crohn’s disease patients, regulates transcriptional programs in macrophages. PHDs crack the histone code Epigenetic readers that recognize histone modifications facilitate histone code–based transcriptional programming. Bromodomain- and plant homeodomain (PHD)–containing proteins often serve as readers of acetylation or methylation on histones, respectively. Mehta et al. have examined the function of SP140, a bromodomain- and PHD domain–containing reader in immune cells, and report that SP140 plays an essential role in repressing expression of lineage-inappropriate genes in macrophages. The authors also propose that SP140 polymorphisms associated with the development of Crohn’s disease represent functional loss of SP140. These studies advance our understanding of how epigenetic readers regulate immune responses in normal and diseased states. Epigenetic “readers” that recognize defined posttranslational modifications on histones have become desirable therapeutic targets for cancer and inflammation. SP140 is one such bromodomain- and plant homeodomain (PHD)–containing reader with immune-restricted expression, and single-nucleotide polymorphisms (SNPs) within SP140 associate with Crohn’s disease (CD). However, the function of SP140 and the consequences of disease-associated SP140 SNPs have remained unclear. We show that SP140 is critical for transcriptional programs that uphold the macrophage state. SP140 preferentially occupies promoters of silenced, lineage-inappropriate genes bearing the histone modification H3K27me3, such as the HOXA cluster in human macrophages, and ensures their repression. Depletion of SP140 in mouse or human macrophages resulted in severely compromised microbe-induced activation. We reveal that peripheral blood mononuclear cells (PBMCs) or B cells from individuals carrying CD-associated SNPs within SP140 have defective SP140 messenger RNA splicing and diminished SP140 protein levels. Moreover, CD patients carrying SP140 SNPs displayed suppressed innate immune gene signatures in a mixed population of PBMCs that stratified them from other CD patients. Hematopoietic-specific knockdown of Sp140 in mice resulted in exacerbated dextran sulfate sodium (DSS)–induced colitis, and low SP140 levels in human CD intestinal biopsies correlated with relatively lower intestinal innate cytokine levels and improved response to anti–tumor necrosis factor (TNF) therapy. Thus, the epigenetic reader SP140 is a key regulator of macrophage transcriptional programs for cellular state, and a loss of SP140 due to genetic variation contributes to a molecularly defined subset of CD characterized by ineffective innate immunity, normally critical for intestinal homeostasis.

Next-Generation Sequencing for Identification of EMS-Induced Mutations in Caenorhabditis elegans.

Forward genetic analysis using chemical mutagenesis in model organisms is a powerful tool for investigation of molecular mechanisms in biological systems. In the nematode, Caenorhabditis elegans, mutagenesis screens using ethyl methanesulfonate (EMS) have led to important insights into genetic control of animal development and physiology. A major bottleneck to this approach is identification of the causative mutation underlying a phenotype of interest. In the past, this has required time‐consuming genetic mapping experiments. More recently, next‐generation sequencing technologies have allowed development of new methods for rapid mapping and identification of EMS‐induced lesions. In this unit we describe a protocol to map and identify EMS‐induced mutations in C. elegans.

The surveillance of pre-mRNA splicing is an early step in C. elegans RNAi of endogenous genes

RNAi pathways detect and silence foreign nucleic acids such as viruses as well as endogenous genes in many species. The phylogenetic profile across eukaryotes of proteins that mediate key steps in RNAi is correlated with the profiles of multiple mRNA splicing proteins and with intron number, suggesting that RNAi may surveil mRNA splicing to detect the divergent or absent introns of viruses. Here we examine the role of mRNA splicing in Caenorhabditis elegans RNAi. We found that viable null mutations in U1 and U2 small nuclear ribonucleic protein (snRNP)-specific splicing factor genes cause defects in RNAi. The U1A ortholog rnp-2 is required for normal ERGO-1 Argonaute class 26G siRNA biogenesis, trans-splicing of the eri-6/7 transcript, and targeting of poorly conserved gene transcripts by WAGO Argonaute class 22G siRNAs. We found that gene transcripts engaged by the siRNA-generating machinery are poorly conserved, possess few introns, and often have introns that are divergent from introns with strong consensus splicing sites found in highly conserved genes. We present biochemical evidence that RNAi targeted transcripts are tightly bound to spliceosomes. These findings suggest multiple layers of regulation by the spliceosome at early steps of small RNA-mediated gene silencing.

Nudt21 Controls Cell Fate by Connecting Alternative Polyadenylation to Chromatin Signaling

Cell fate transitions involve rapid gene expression changes and global chromatin remodeling, yet the underlying regulatory pathways remain incompletely understood. Here, we identified the RNA-processing factor Nudt21 as a novel regulator of cell fate change using transcription-factor-induced reprogramming as a screening assay. Suppression of Nudt21 enhanced the generation of induced pluripotent stem cells, facilitated transdifferentiation into trophoblast stem cells, and impaired differentiation of myeloid precursors and embryonic stem cells, suggesting a broader role for Nudt21 in cell fate change. We show that Nudt21 directs differential polyadenylation of over 1,500 transcripts in cells acquiring pluripotency, although only a fraction changed protein levels. Remarkably, these proteins were strongly enriched for chromatin regulators, and their suppression neutralized the effect of Nudt21 during reprogramming. Collectively, our data uncover Nudt21 as a novel post-transcriptional regulator of cell fate and establish a direct, previously unappreciated link between alternative polyadenylation and chromatin signaling.

Synergistic activity and heterogeneous acquired resistance of combined MDM2 and MEK inhibition in KRAS mutant cancers

There are currently no effective targeted therapies for KRAS mutant cancers. Therapeutic strategies that combine MEK inhibitors with agents that target apoptotic pathways may be a promising therapeutic approach. We investigated combining MEK and MDM2 inhibitors as a potential treatment strategy for KRAS mutant non-small cell lung cancers (NSCLC) and colorectal carcinomas that harbor wild-type TP53. The combination of pimasertib (MEK inhibitor) and SAR405838 (MDM2 inhibitor) was synergistic and induced the expression of PUMA and BIM, led to apoptosis and growth inhibition in vitro, and tumor regression in vivo. Acquired resistance to the combination commonly resulted from the acquisition of TP53 mutations, conferring complete resistance to MDM2 inhibition. In contrast, resistant clones exhibited marked variability in sensitivity to MEK inhibition, which significantly impacted sensitivity to subsequent treatment with alternative MEK inhibitor-based combination therapies. These results highlight both the potential promise and limitations of combining MEK and MDM2 inhibitors for treatment of KRAS mutant NSCLC and colorectal cancers.

Mutant GNAS drives pancreatic tumourigenesis by inducing PKA-mediated SIK suppression and reprogramming lipid metabolism

G protein αs (GNAS) mediates receptor-stimulated cAMP signalling, which integrates diverse environmental cues with intracellular responses. GNAS is mutationally activated in multiple tumour types, although its oncogenic mechanisms remain elusive. We explored this question in pancreatic tumourigenesis where concurrent GNAS and KRAS mutations characterize pancreatic ductal adenocarcinomas (PDAs) arising from intraductal papillary mucinous neoplasms (IPMNs). By developing genetically engineered mouse models, we show that GnasR201C cooperates with KrasG12D to promote initiation of IPMN, which progress to invasive PDA following Tp53 loss. Mutant Gnas remains critical for tumour maintenance in vivo. This is driven by protein-kinase-A-mediated suppression of salt-inducible kinases (Sik1–3), associated with induction of lipid remodelling and fatty acid oxidation. Comparison of Kras-mutant pancreatic cancer cells with and without Gnas mutations reveals striking differences in the functions of this network. Thus, we uncover Gnas-driven oncogenic mechanisms, identify Siks as potent tumour suppressors, and demonstrate unanticipated metabolic heterogeneity among Kras-mutant pancreatic neoplasms.Bardeesy and colleagues show that mutant GNAS suppresses salt-inducible kinases by activating PKA, leading to lipid remodelling and pancreatic tumourigenesis