Scientific Papers

For more papers, visit a faculty member's page from the listing on Whitehead Faculty and access the PubMed link.

NUFIP1 is a ribosome receptor for starvation-induced ribophagy.

Science. 2018 May 18;360(6390):751-758. 

Wyant, G.A.*, Abu-Remaileh, M.*, Frenkel, E.M.*, Laqtom, N.N.*, Dharamdasani, V.*, Lewis, C.A.*, Chan, S.H.*, Heinze, I., Ori, A., and Sabatini, D.M.

The lysosome degrades and recycles macromolecules, signals to the master growth regulator mTORC1, and is associated with human disease. Here, we performed quantitative proteomic analyses of lysosomes rapidly isolated using the LysoIP method and find that nutrient levels and mTOR dynamically modulate the lysosomal proteome. We focus on NUFIP1, a protein that upon mTORC1 inhibition redistributes from the nucleus to autophagosomes and lysosomes. Upon these conditions, NUFIP1 interacts with ribosomes and delivers them to autophagosomes by directly binding to LC3B. The starvation-induced degradation of ribosomes via autophagy (ribophagy) depends on the capacity of NUFIP1 to bind LC3B and promotes cell survival. We propose that NUFIP1 is a receptor for the selective autophagy of ribosomes.

 

Understanding the tumor immune microenvironment (TIME) for effective therapy.

Nat Med. 2018 May;24(5):541-550.

Binnewies, M., Roberts, E.W., Kersten, K., Chan, V., Fearon, D.F., Merad, M., Coussens, L.M., Gabrilovich, D.I., Ostrand-Rosenberg, S., Hedrick, C.C., Vonderheide, R.H., Pittet, M.J., Jain, R.K., Zou, W., Howcroft, T.K., Woodhouse, E.C., Weinberg, R.A.*, and Krummel, M.F.

The clinical successes in immunotherapy have been both astounding and at the same time unsatisfactory. Countless patients with varied tumor types have seen pronounced clinical response with immunotherapeutic intervention; however, many more patients have experienced minimal or no clinical benefit when provided the same treatment. As technology has advanced, so has the understanding of the complexity and diversity of the immune context of the tumor microenvironment and its influence on response to therapy. It has been possible to identify different subclasses of immune environment that have an influence on tumor initiation and response and therapy; by parsing the unique classes and subclasses of tumor immune microenvironment (TIME) that exist within a patient's tumor, the ability to predict and guide immunotherapeutic responsiveness will improve, and new therapeutic targets will be revealed.

 

Enhanced phosphocholine metabolism is essential for terminal erythropoiesis.

Blood. 2018 Apr 30.

Huang, N.J.*, Lin, Y.C., Lin, C.Y., Pishesha, N., Lewis, C.A.*, Freinkman, E.*, Farquharson, C., Millan, J.L., and Lodish, H.*

Red cells contain a unique constellation of membrane lipids. While much is known about regulated protein expression, the regulation of lipid metabolism during erythropoiesis is poorly studied. Here we show that transcription of PHOSPHO1, a phosphoethanolamine and phosphocholine phosphatase that mediates the hydrolysis of phosphocholine to choline, is strongly upregulated during the terminal stages of erythropoiesis of both human and mouse erythropoiesis, concomitant with increased catabolism of phosphatidylcholine and phosphocholine as shown by global lipidomic analyses of mouse and human terminal erythropoiesis. Depletion of PHOSPHO1 impaired differentiation of fetal mouse and human erythroblasts, and in adult mice depletion impaired phenylhydrazine-induced stress erythropoiesis. Loss of PHOSPHO1 also impaired phosphocholine catabolism in mouse fetal liver progenitors and resulted in accumulation of several lipids; ATP production was reduced as a result of decreased oxidative phosphorylation. Glycolysis replaced oxidative phosphorylation in PHOSPHO1 knockout erythroblasts and the increased glycolysis was used for the production of serine or glycine. Our study elucidates the dynamic changes in lipid metabolism during terminal erythropoiesis and reveals the key roles of phosphatidylcholine and phosphocholine metabolism in energy balance and amino acid supply.

 

New CRISPR mutagenesis strategies reveal variation in repair mechanisms among fungi.

mSphere. 2018 Apr 25;3(2).

Vyas, V.K.*, Bushkin, G.G.*, Bernstein, D.A.*, Getz, M.A.*, Sewastianik, M.*, Barrasa, M.I.*, Bartel, D.P.*, and Fink, G.R.*

We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii. These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms.IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata, as well as in the related yeasts S. cerevisiae and N. castellii. The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.

 

Cell type transcriptome atlas for the planarian Schmidtea mediterranea.

Science. 2018 Apr 19.

Fincher, C.T.*, Wurtzel, O.*, de Hoog, T.*, Kravarik, K.M.*, and Reddien, P.W.*

The transcriptome of a cell dictates its unique cell-type biology. We used single-cell RNA sequencing to determine the transcriptomes for essentially every cell type of a complete animal: the regenerative planarian Schmidtea mediterranea. Planarians contain a diverse array of cell types, possess lineage progenitors for differentiated cells (including pluripotent stem cells), and constitutively express positional information, making them ideal for this undertaking. We generated data for 66,783 cells, defining transcriptomes for known and many previously unknown planarian cell types and for putative transition states between stem and differentiated cells. We also uncovered regionally expressed genes in muscle, which harbors positional information. Identifying the transcriptomes for potentially all cell types for many organisms should be readily attainable and is a powerful new approach to metazoan biology.

 

A genetic program mediates cold-warming response and promotes stress-induced phenoptosis in C. elegans.

Elife. 2018 Apr 17;7. 

Jiang, W., Wei, Y., Long, Y., Owen, A., Wang, B., Wu, X., Luo, S.*, Dang, Y., and Ma, D.K.

How multicellular organisms respond to and are impacted by severe hypothermic stress is largely unknown. From C. elegans screens for mutants abnormally responding to cold-warming stimuli, we identify a molecular genetic pathway comprising ISY-1, a conserved uncharacterized protein, and ZIP-10, a bZIP-type transcription factor. ISY-1 gatekeeps the ZIP-10 transcriptional program by regulating the microRNA mir-60. Downstream of ISY-1 and mir-60, zip-10 levels rapidly and specifically increase upon transient cold-warming exposure. Prolonged zip-10 up-regulation induces several protease-encoding genes and promotes stress-induced organismic death, or phenoptosis, of C. elegans. zip-10 deficiency confers enhanced resistance to prolonged cold-warming stress, more prominently in adults than larvae. We conclude that the ZIP-10 genetic program mediates cold-warming response and may have evolved to promote wild-population kin selection under resource-limiting and thermal stress conditions.

 

Ablation of insulin receptor substrates 1 and 2 suppresses Kras-driven lung tumorigenesis.

Proc Natl Acad Sci U S A. 2018 Apr 17;115(16):4228-4233.

Xu, H., Lee, M.S., Tsai, P.Y., Adler, A.S., Curry, N.L., Challa, S., Freinkman, E.*, Hitchcock, D.S., Copps, K.D., White, M.F., Bronson, R.T., Marcotrigiano, M., Wu, Y., Clish, C.B., and Kalaany, N.Y.

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer death worldwide, with 25% of cases harboring oncogenic Kirsten rat sarcoma (KRAS). Although KRAS direct binding to and activation of PI3K is required for KRAS-driven lung tumorigenesis, the contribution of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) in the context of mutant KRAS remains controversial. Here, we provide genetic evidence that lung-specific dual ablation of insulin receptor substrates 1/2 (Irs1/Irs2), which mediate insulin and IGF1 signaling, strongly suppresses tumor initiation and dramatically extends the survival of a mouse model of lung cancer with Kras activation and p53 loss. Mice with Irs1/Irs2 loss eventually succumb to tumor burden, with tumor cells displaying suppressed Akt activation and strikingly diminished intracellular levels of essential amino acids. Acute loss of IRS1/IRS2 or inhibition of IR/IGF1R in KRAS-mutant human NSCLC cells decreases the uptake and lowers the intracellular levels of amino acids, while enhancing basal autophagy and sensitivity to autophagy and proteasome inhibitors. These findings demonstrate that insulin/IGF1 signaling is required for KRAS-mutant lung cancer initiation, and identify decreased amino acid levels as a metabolic vulnerability in tumor cells with IR/IGF1R inhibition. Consequently, combinatorial targeting of IR/IGF1R with autophagy or proteasome inhibitors may represent an effective therapeutic strategy in KRAS-mutant NSCLC.

  

The systemic response to surgery triggers the outgrowth of distant immune-controlled tumors in mouse models of dormancy.

Sci Transl Med. 2018 Apr 11;10(436).

Krall, J.A.*, Reinhardt, F.*, Mercury, O.A.*, Pattabiraman, D.R.*, Brooks, M.W.*, Dougan, M.*, Lambert, A.W.*, Bierie, B.*, Ploegh, H.L.*, Dougan, S.K.*, and Weinberg, R.A.*

Patients undergoing surgical resection of primary breast tumors confront a risk for metastatic recurrence that peaks sharply 12 to 18 months after surgery. The cause of early metastatic relapse in breast cancer has long been debated, with many ascribing these relapses to the natural progression of the disease. Others have proposed that some aspect of surgical tumor resection triggers the outgrowth of otherwise-dormant metastases, leading to the synchronous pattern of relapse. Clinical data cannot distinguish between these hypotheses, and previous experimental approaches have not provided clear answers. Such uncertainty hinders the development and application of therapeutic approaches that could potentially reduce early metastatic relapse. We describe an experimental model system that definitively links surgery and the subsequent wound-healing response to the outgrowth of tumor cells at distant anatomical sites. Specifically, we find that the systemic inflammatory response induced after surgery promotes the emergence of tumors whose growth was otherwise restricted by a tumor-specific T cell response. Furthermore, we demonstrate that perioperative anti-inflammatory treatment markedly reduces tumor outgrowth in this model, suggesting that similar approaches might substantially reduce early metastatic recurrence in breast cancer patients.

 

Transcriptional dysregulation of MYC reveals common enhancer-docking mechanism.

Cell Rep. 2018 Apr 10;23(2):349-360.

Schuijers, J.*, Manteiga, J.C.*, Weintraub, A.S.*, Day, D.S.*, Zamudio, A.V.*, Hnisz, D.*, Lee, T.I.*, and Young, R.A.*

Transcriptional dysregulation of the MYC oncogene is among the most frequent events in aggressive tumor cells, and this is generally accomplished by acquisition of a super-enhancer somewhere within the 2.8 Mb TAD where MYC resides. We find that these diverse cancer-specific super-enhancers, differing in size and location, interact with the MYC gene through a common and conserved CTCF binding site located 2 kb upstream of the MYC promoter. Genetic perturbation of this enhancer-docking site in tumor cells reduces CTCF binding, super-enhancer interaction, MYC gene expression, and cell proliferation. CTCF binding is highly sensitive to DNA methylation, and this enhancer-docking site, which is hypomethylated in diverse cancers, can be inactivated through epigenetic editing with dCas9-DNMT. Similar enhancer-docking sites occur at other genes, including genes with prominent roles in multiple cancers, suggesting a mechanism by which tumor cell oncogenes can generally hijack enhancers. These results provide insights into mechanisms that allow a single target gene to be regulated by diverse enhancer elements in different cell types.

 

Toxoplasma CRISPR/Cas9 constructs are functional for gene disruption in Neospora caninum.

Int J Parasitol. 2018 Apr 3.

Arranz-Solis, D., Regidor-Cerrillo, J., Lourido, S.*, Ortega-Mora, L.M., and Saeij, J.P.J.

Herein we describe, to our knowledge for the first time the use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 (CRISPR/Cas9) system for genome editing of Neospora caninum, an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide. By using plasmids containing the CRISPR/Cas9 components adapted to the closely related parasite Toxoplasma gondii, we successfully knocked out a green fluorescent protein (GFP) in an Nc-1 GFP-expressing strain, and efficiently disrupted the NcGRA7 gene in the Nc-Spain7 isolate by insertion of a pyrimethamine resistance cassette. The successful use of this technology in N. caninum lays the foundation for an efficient, targeted gene modification tool in this parasite. 


 

*Author affiliated with Whitehead Institute for Biomedical Research

© Whitehead Institute for Biomedical Research              455 Main Street          Cambridge, MA 02142