Scientific Papers

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

Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors.

Nat Chem Biol. 2016 Aug 29.

Zhang, T., Kwiatkowski, N.*, Olson, C.M., Dixon-Clarke, S.E., Abraham, B.J.*, Greifenberg, A.K., Ficarro, S.B., Elkins, J.M., Liang, Y., Hannett, N.M.*, Manz, T., Hao, M., Bartkowiak, B, Greenleaf, A.L., Marto, J.A., Geyer, M., Bullock, A.N., Young, R.A.*, and Gray, N.S.

Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.

 

Cell-type-specific alternative splicing governs cell fate in the developing cerebral cortex.

Cell. 2016 Aug 25;166(5):1147-1162.e15.

Zhang, X., Chen, M.H., Wu, X.*, Kodani, A., Fan, J., Doan, R., Ozawa, M., Ma, J., Yoshida, N., Reiter, J.F., Black, D.L., Kharchenko, P.V., Sharp, P.A., and Walsh, C.A.

Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains-especially in cytoskeletal proteins-and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development.

 

Absolute quantification of matrix metabolites reveals the dynamics of mitochondrial metabolism.

Cell. 2016 Aug 25;166(5):1324-1337.e11

Chen, W.W.*, Freinkman, E.*, Wang, T.*, Birsoy, K., and Sabatini, D.M.*

Mitochondria house metabolic pathways that impact most aspects of cellular physiology. While metabolite profiling by mass spectrometry is widely applied at the whole-cell level, it is not routinely possible to measure the concentrations of small molecules in mammalian organelles. We describe a method for the rapid and specific isolation of mitochondria and use it in tandem with a database of predicted mitochondrial metabolites ("MITObolome") to measure the matrix concentrations of more than 100 metabolites across various states of respiratory chain (RC) function. Disruption of the RC reveals extensive compartmentalization of mitochondrial metabolism and signatures unique to the inhibition of each RC complex. Pyruvate enables the proliferation of RC-deficient cells but has surprisingly limited effects on matrix contents. Interestingly, despite failing to restore matrix NADH/NAD balance, pyruvate does increase aspartate, likely through the exchange of matrix glutamate for cytosolic aspartate. We demonstrate the value of mitochondrial metabolite profiling and describe a strategy applicable to other organelles.

 

Posttranscriptional regulation of glycoprotein quality control in the endoplasmic reticulum is controlled by the E2 Ub-conjugating enzyme UBC6e.

Mol Cell. 2016 Aug 24.

Hagiwara, M.*, Ling, J.*, Koenig, P.A.*, and Ploegh, H.L.*

ER-associated degradation (ERAD) is essential for protein quality control in the ER, not only when the ER is stressed, but also at steady state. We report a new layer of homeostatic control, in which ERAD activity itself is regulated posttranscriptionally and independently of the unfolded protein response by adjusting the endogenous levels of EDEM1, OS-9, and SEL1L (ERAD enhancers). Functional UBC6e requires its precise location in the ER to form a supramolecular complex with Derlin2. This complex targets ERAD enhancers for degradation, a function that depends on UBC6e's enzymatic activity. Ablation of UBC6e causes upregulation of active ERAD enhancers and so increases clearance not only of terminally misfolded substrates, but also of wild-type glycoproteins that fold comparatively slowly in vitro and in vivo. The levels of proteins that comprise the ERAD machinery are thus carefully tuned and adjusted to prevailing needs.

 

Recurrent somatic mutations in POLR2A define a distinct subset of meningiomas.

Nat Genet. 2016 Aug 22.

Clark, V.E., Harmanci, A.S., Bai, H., Youngblood, M.W., Lee, T.I.*, Baranoski, J.F., Ercan-Sencicek, A.G., Abraham, B.J.*, Weintraub, A.S.,* Hnisz, D.*, Simon, M., Krischek, B., Erson-Omay, E.Z., Henegariu, O., Carrión-Grant, G., Mishra-Gorur, K., Durán, D., Goldmann, J.E.*, Schramm, J., Goldbrunner, R., Piepmeier, J.M., Vortmeyer, A.O., Günel, J.M., Bilgüvar, K., Yasuno, K., Young, R.A.*, and Günel, M.

RNA polymerase II mediates the transcription of all protein-coding genes in eukaryotic cells, a process that is fundamental to life. Genomic mutations altering this enzyme have not previously been linked to any pathology in humans, which is a testament to its indispensable role in cell biology. On the basis of a combination of next-generation genomic analyses of 775 meningiomas, we report that recurrent somatic p.Gln403Lys or p.Leu438_His439del mutations in POLR2A, which encodes the catalytic subunit of RNA polymerase II (ref. 1), hijack this essential enzyme and drive neoplasia. POLR2A mutant tumors show dysregulation of key meningeal identity genes, including WNT6 and ZIC1/ZIC4. In addition to mutations in POLR2A, NF2, SMARCB1, TRAF7, KLF4, AKT1, PIK3CA, and SMO, we also report somatic mutations in AKT3, PIK3R1, PRKAR1A, and SUFU in meningiomas. Our results identify a role for essential transcriptional machinery in driving tumorigenesis and define mutually exclusive meningioma subgroups with distinct clinical and pathological features.

 

Validation of protein knockout in mutant zebrafish lines using in vitro translation assays.

Zebrafish. 2016 Aug 22.

Carter, B.S.*, Cortes-Campos, C.*, Chen, X., McCammon, J.M.*, and Sive, H.L.*

Advances in genome-editing technology have made creation of zebrafish mutant lines accessible to the community. Experimental validation of protein knockout is a critical step in verifying null mutants, but this can be a difficult task. Absence of protein can be confirmed by Western blotting; however, this approach requires target-specific antibodies that are generally not available for zebrafish proteins. We address this issue using in vitro translation assays, a fast and standard procedure that can be easily implemented.

 

Inflammation triggers Zeb1-dependent escape from tumor latency.

Cancer Res. 2016 Aug 16.

De Cock, J.M.*, Shibue, T.*, Dongre, A.*, Keckesova, Z.*, Reinhardt, F.*, and Weinberg, R.A.*

The emergence of metastatic disease in cancer patients many years or decades after initial successful treatment of primary tumors is well documented but poorly understood at the molecular level. Recent studies have begun exploring the cell-intrinsic programs causing disseminated tumor cells to enter latency and the cellular signals in the surrounding non-permissive tissue microenvironment that maintain the latent state. However, relatively little is known about the mechanisms that enable disseminated tumor cells to escape cancer dormancy or tumor latency. We describe here an in vivo model of solitary metastatic latency in the lung parenchyma. The induction of a localized inflammation in the lungs, initiated by lipopolysaccharide (LPS) treatment, triggers the awakening of these cells, which develop into macroscopic metastases. The escape from latency is dependent on the expression of Zeb1, a key regulator of the epithelial-to-mesenchymal transition (EMT). Furthermore, activation of the EMT program on its own, as orchestrated by Zeb1, is sufficient to incite metastatic outgrowth by causing carcinoma cells to enter stably into a metastasis-initiating cell state.

 

A fungal-selective cytochrome bc1 inhibitor impairs virulence and prevents the evolution of drug resistance.

Cell Chem Biol. 2016 Aug 6.

Vincent, B.M.*, Langlois, J.B., Srinivas, R., Lancaster, A.K.*, Scherz-Shouval, R.*, Whitesell, L*., Tidor, B., Buchwald, S.L., and Lindquist, S.*

To cause disease, a microbial pathogen must adapt to the challenges of its host environment. The leading fungal pathogen Candida albicans colonizes nutrient-poor bodily niches, withstands attack from the immune system, and tolerates treatment with azole antifungals, often evolving resistance. To discover agents that block these adaptive strategies, we screened 300,000 compounds for inhibition of azole tolerance in a drug-resistant Candida isolate. We identified a novel indazole derivative that converts azoles from fungistatic to fungicidal drugs by selective inhibition of mitochondrial cytochrome bc1. We synthesized 103 analogs to optimize potency (half maximal inhibitory concentration 0.4 muM) and fungal selectivity (28-fold over human). In addition to reducing azole resistance, targeting cytochrome bc1 prevents C. albicans from adapting to the nutrient-deprived macrophage phagosome and greatly curtails its virulence in mice. Inhibiting mitochondrial respiration and restricting metabolic flexibility with this synthetically tractable chemotype provides an attractive therapeutic strategy to limit both fungal virulence and drug resistance.

 

Phenotypic lentivirus screens to identify functional single domain antibodies.

Nat Microbiol. 2016 Jun 20;1(8):16080.

Schmidt, F.I.*, Hanke, L.*, Morin, B., Brewer, R.*, Brusic, V., Whelan, S.P., and Ploegh, H.L.*

Manipulation of proteins is key in assessing their in vivo function. Although genetic ablation is straightforward, reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as camelid-derived VHHs, can serve as inhibitors or activators of intracellular protein function, but functional testing of identified VHHs is laborious. To address this challenge, we have developed a lentiviral screening approach to identify VHHs that elicit a phenotype when expressed intracellularly. We identified 19 antiviral VHHs that protect human A549 cells from lethal infection with influenza A virus (IAV) or vesicular stomatitis virus (VSV), respectively. Both negative-sense RNA viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins. Antiviral VHHs prevented nuclear import of viral ribonucleoproteins or mRNA transcription, respectively, and may provide clues for novel antiviral reagents. In principle, the screening approach described here should be applicable to identify inhibitors of any pathogen or biological pathway.

 

The combined deficiency of immunoproteasome subunits affects both the magnitude and quality of pathogen- and genetic vaccination-induced CD8(+) T cell responses to the human protozoan parasite Trypanosoma cruzi.

PLoS Pathog. 2016 Apr 29;12(4):e1005593.

Ersching, J.*, Vasconcelos, J.R., Ferreira, C.P., Caetano, B.C., Machado, A.V., Bruna-Romero, O., Baron, M.A., Ferreira, L.R.P., Cunha-Neto, E., Rock, K.L., Gazzinelli, R.T., and Rodrigues, M.M.

The beta 1i, beta 2i and beta 5i immunoproteasome subunits have an important role in defining the repertoire of MHC class I-restricted epitopes. However, the impact of combined deficiency of the three immunoproteasome subunits in the development of protective immunity to intracellular pathogens has not been investigated. Here, we demonstrate that immunoproteasomes play a key role in host resistance and genetic vaccination-induced protection against the human pathogen Trypanosoma cruzi (the causative agent of Chagas disease), immunity to which is dependent on CD8(+) T cells and IFN-gamma (the classical immunoproteasome inducer). We observed that infection with T. cruzi triggers the transcription of immunoproteasome genes, both in mice and humans. Importantly, genetically vaccinated or T. cruzi infected beta 1i, beta 2i and beta 5i triple knockout (TKO) mice presented significantly lower frequencies and numbers of splenic CD8(+) effector T cells (CD8(+) CD44(high)CD62L(low)) specific for the previously characterized immunodominant (VNHRFTLV) H-2K(b)-restricted T. cruzi epitope. Not only the quantity, but also the quality of parasite-specific CD8(+) T cell responses was altered in TKO mice. Hence, the frequency of double-positive (IFN-gamma(+)/TNF+) or single-positive (IFN-gamma(+)) cells specific for the H-2K(b)-restricted immunodominant as well as subdominant T. cruzi epitopes were higher inWT mice, whereas TNF single-positive cells prevailed among CD8(+) T cells from TKO mice. Contrasting with their WT counterparts, TKO animals were also lethally susceptible to T. cruzi challenge, even after an otherwise protective vaccination with DNA and adenoviral vectors. We conclude that the immunoproteasome subunits are key determinants in host resistance to T. cruzi infection by influencing both the magnitude and quality of CD8(+) T cell responses.

 

*Author affiliated with Whitehead Institute for Biomedical Research

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