Publications by authors named "Kevan M Shokat"

294 Publications

Phospholipidosis is a shared mechanism underlying the antiviral activity of many repurposed drugs against SARS-CoV-2.

bioRxiv 2021 Mar 24. Epub 2021 Mar 24.

Repurposing drugs as treatments for COVID-19 has drawn much attention. A common strategy has been to screen for established drugs, typically developed for other indications, that are antiviral in cells or organisms. Intriguingly, most of the drugs that have emerged from these campaigns, though diverse in structure, share a common physical property: cationic amphiphilicity. Provoked by the similarity of these repurposed drugs to those inducing phospholipidosis, a well-known drug side effect, we investigated phospholipidosis as a mechanism for antiviral activity. We tested 23 cationic amphiphilic drugs-including those from phenotypic screens and others that we ourselves had found-for induction of phospholipidosis in cell culture. We found that most of the repurposed drugs, which included hydroxychloroquine, azithromycin, amiodarone, and four others that have already progressed to clinical trials, induced phospholipidosis in the same concentration range as their antiviral activity; indeed, there was a strong monotonic correlation between antiviral efficacy and the magnitude of the phospholipidosis. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the gross physical properties of drugs, and does not reflect specific target-based activities, rather it may be considered a confound in early drug discovery. Understanding its role in infection, and detecting its effects rapidly, will allow the community to better distinguish between drugs and lead compounds that more directly impact COVID-19 from the large proportion of molecules that manifest this confounding effect, saving much time, effort and cost.

One Sentence Summary: Drug-induced phospholipidosis is a single mechanism that may explain the efficacy of a wide-variety of therapeutics repurposed for COVID-19.
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http://dx.doi.org/10.1101/2021.03.23.436648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010720PMC
March 2021

Drugging the "Undruggable" MYCN Oncogenic Transcription Factor: Overcoming Previous Obstacles to Impact Childhood Cancers.

Cancer Res 2021 Apr 28;81(7):1627-1632. Epub 2021 Jan 28.

Division of Oncology and Center for Childhood Cancer Research, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

Effective treatment of pediatric solid tumors has been hampered by the predominance of currently "undruggable" driver transcription factors. Improving outcomes while decreasing the toxicity of treatment necessitates the development of novel agents that can directly inhibit or degrade these elusive targets. MYCN in pediatric neural-derived tumors, including neuroblastoma and medulloblastoma, is a paradigmatic example of this problem. Attempts to directly and specifically target MYCN have failed due to its similarity to MYC, the unstructured nature of MYC family proteins in their monomeric form, the lack of an understanding of MYCN-interacting proteins and ability to test their relevance , the inability to obtain structural information on MYCN protein complexes, and the challenges of using traditional small molecules to inhibit protein-protein or protein-DNA interactions. However, there is now promise for directly targeting MYCN based on scientific and technological advances on all of these fronts. Here, we discuss prior challenges and the reasons for renewed optimism in directly targeting this "undruggable" transcription factor, which we hope will lead to improved outcomes for patients with pediatric cancer and create a framework for targeting driver oncoproteins regulating gene transcription.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-3108DOI Listing
April 2021

Host-directed therapies against early-lineage SARS-CoV-2 retain efficacy against B.1.1.7 variant.

bioRxiv 2021 Jan 24. Epub 2021 Jan 24.

Quantitative Biosciences Institute (QBI) Coronavirus Research Group (QCRG), San Francisco, CA 94158, USA.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths worldwide and massive societal and economic burden. Recently, a new variant of SARS-CoV-2, known as B.1.1.7, was first detected in the United Kingdom and is spreading in several other countries, heightening public health concern and raising questions as to the resulting effectiveness of vaccines and therapeutic interventions. We and others previously identified host-directed therapies with antiviral efficacy against SARS-CoV-2 infection. Less prone to the development of therapy resistance, host-directed drugs represent promising therapeutic options to combat emerging viral variants as host genes possess a lower propensity to mutate compared to viral genes. Here, in the first study of the full-length B.1.1.7 variant virus, we find two host-directed drugs, plitidepsin (aplidin; inhibits translation elongation factor eEF1A) and ralimetinib (inhibits p38 MAP kinase cascade), as well as remdesivir, to possess similar antiviral activity against both the early-lineage SARS-CoV-2 and the B.1.1.7 variant, evaluated in both human gastrointestinal and lung epithelial cell lines. We find that plitidepsin is over an order of magnitude more potent than remdesivir against both viruses. These results highlight the importance of continued development of host-directed therapeutics to combat current and future coronavirus variant outbreaks.
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http://dx.doi.org/10.1101/2021.01.24.427991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836107PMC
January 2021

Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A.

Science 2021 02 25;371(6532):926-931. Epub 2021 Jan 25.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.
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http://dx.doi.org/10.1126/science.abf4058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7963220PMC
February 2021

GTP-State-Selective Cyclic Peptide Ligands of K-Ras(G12D) Block Its Interaction with Raf.

ACS Cent Sci 2020 Oct 23;6(10):1753-1761. Epub 2020 Sep 23.

Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

We report the identification of three cyclic peptide ligands of K-Ras(G12D) using an integrated translation-mRNA display selection platform. These cyclic peptides show preferential binding to the GTP-bound state of K-Ras(G12D) over the GDP-bound state and block Ras-Raf interaction. A co-crystal structure of peptide KD2 with K-Ras(G12D)·GppNHp reveals that this peptide binds in the Switch II groove region with concomitant opening of the Switch II loop and a 40° rotation of the α2 helix, and that a threonine residue (Thr10) on KD2 has direct access to the mutant aspartate (Asp12) on K-Ras. Replacing this threonine with non-natural amino acids afforded peptides with improved potency at inhibiting the interaction between Raf1-RBD and K-Ras(G12D) but not wildtype K-Ras. The union of G12D over wildtype selectivity and GTP state/GDP state selectivity is particularly desirable, considering that oncogenic K-Ras(G12D) exists predominantly in the GTP state in cancer cells, and wildtype K-Ras signaling is important for the maintenance of healthy cells.
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http://dx.doi.org/10.1021/acscentsci.0c00514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596874PMC
October 2020

The splicing modulator sulfonamide indisulam reduces AR-V7 in prostate cancer cells.

Bioorg Med Chem 2020 10 18;28(20):115712. Epub 2020 Aug 18.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Alternative splicing of the androgen receptor (AR) is frequently observed in castration resistant prostate cancer (CRPC). One AR isoform, the AR-V7 splice variant, is a constitutively active transcription factor which lacks a ligand binding domain and is therefore undruggable. AR-V7 expression correlates with resistance to androgen receptor signaling inhibitors (ARSi) and poor clinical prognoses. The occurrence of the AR-V7 splice variant is driven by alternative splicing of AR pre-mRNA by the spliceosome, however the mechanistic details are poorly understood. We demonstrate that the splicing factor RBM39 is critical for alternative splicing of the AR-V7 splice variant mRNA transcripts from AR pre-mRNA, and that the anti-cancer drug, indisulam, reduces AR-V7 mRNA levels by degrading RBM39. We report that indisulam effectively reduces AR-V7 in in vitro and in vivo models.
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http://dx.doi.org/10.1016/j.bmc.2020.115712DOI Listing
October 2020

Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms.

Science 2020 12 15;370(6521). Epub 2020 Oct 15.

Quantitative Biosciences Institute (QBI) COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study.
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http://dx.doi.org/10.1126/science.abe9403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808408PMC
December 2020

The Global Phosphorylation Landscape of SARS-CoV-2 Infection.

Cell 2020 08 28;182(3):685-712.e19. Epub 2020 Jun 28.

Institute for Clinical and Experimental Pharmacology and Toxicology, University of Freiburg, Freiburg 79104, Germany.

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.
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http://dx.doi.org/10.1016/j.cell.2020.06.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321036PMC
August 2020

Unbiased Proteomic Profiling Uncovers a Targetable GNAS/PKA/PP2A Axis in Small Cell Lung Cancer Stem Cells.

Cancer Cell 2020 07 11;38(1):129-143.e7. Epub 2020 Jun 11.

Department of Pediatrics, Stanford University, 265 Campus Drive, Stanford, CA 94305-5457, USA; Department of Genetics, Stanford University, Stanford, CA 94305, USA. Electronic address:

Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.
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http://dx.doi.org/10.1016/j.ccell.2020.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363571PMC
July 2020

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.

Nature 2020 07 30;583(7816):459-468. Epub 2020 Apr 30.

QBI COVID-19 Research Group (QCRG), San Francisco, CA, USA.

A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.
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http://dx.doi.org/10.1038/s41586-020-2286-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431030PMC
July 2020

p27 allosterically activates cyclin-dependent kinase 4 and antagonizes palbociclib inhibition.

Science 2019 12;366(6471)

Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA.

The p27 protein is a canonical negative regulator of cell proliferation and acts primarily by inhibiting cyclin-dependent kinases (CDKs). Under some circumstances, p27 is associated with active CDK4, but no mechanism for activation has been described. We found that p27, when phosphorylated by tyrosine kinases, allosterically activated CDK4 in complex with cyclin D1 (CDK4-CycD1). Structural and biochemical data revealed that binding of phosphorylated p27 (phosp27) to CDK4 altered the kinase adenosine triphosphate site to promote phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and other substrates. Surprisingly, purified and endogenous phosp27-CDK4-CycD1 complexes were insensitive to the CDK4-targeting drug palbociclib. Palbociclib instead primarily targeted monomeric CDK4 and CDK6 (CDK4/6) in breast tumor cells. Our data characterize phosp27-CDK4-CycD1 as an active Rb kinase that is refractory to clinically relevant CDK4/6 inhibitors.
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http://dx.doi.org/10.1126/science.aaw2106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7592119PMC
December 2019

Cooperative Blockade of PKCα and JAK2 Drives Apoptosis in Glioblastoma.

Cancer Res 2020 02 5;80(4):709-718. Epub 2019 Dec 5.

Department of Neurology, University of California, San Francisco, California.

The mTOR signaling is dysregulated prominently in human cancers including glioblastoma, suggesting mTOR as a robust target for therapy. Inhibitors of mTOR have had limited success clinically, however, in part because their mechanism of action is cytostatic rather than cytotoxic. Here, we tested three distinct mTOR kinase inhibitors (TORKi) PP242, KU-0063794, and sapanisertib against glioblastoma cells. All agents similarly decreased proliferation of glioblastoma cells, whereas PP242 uniquely induced apoptosis. Apoptosis induced by PP242 resulted from off-target cooperative inhibition of JAK2 and protein kinase C alpha (PKCα). Induction of apoptosis was also decreased by additional on-target inhibition of mTOR, due to induction of autophagy. As EGFR inhibitors can block PKCα, EGFR inhibitors erlotinib and osimertinib were tested separately in combination with the JAK2 inhibitor AZD1480. Combination therapy induced apoptosis of glioblastoma tumors in both flank and in patient-derived orthotopic xenograft models, providing a preclinical rationale to test analogous combinations in patients. SIGNIFICANCE: These findings identify PKCα and JAK2 as targets that drive apoptosis in glioblastoma, potentially representing a clinically translatable approach for glioblastoma.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-2808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7024655PMC
February 2020

Betacellulin drives therapy resistance in glioblastoma.

Neuro Oncol 2020 04;22(4):457-469

Department of Neurology, University of California San Francisco (UCSF), San Francisco, California.

Background: The transcription factor signal transducer and activator of transcription 3 (STAT3) drives progression in glioblastoma (GBM), suggesting STAT3 as a therapeutic target. Surprisingly however, GBM cells generally show primary resistance to STAT3 blockade.

Methods: Human glioblastoma cell lines LN229, U87, SF767, and U373, and patient-derived xenografts (PDXs) GBM8 and GBM43 were used to evaluate epidermal growth factor receptor (EGFR) activation during STAT3 inhibition. Protein and gene expression experiments, protein stability assays, cytokine arrays, phospho-tyrosine arrays and EGFR-ligand protein arrays were performed on STAT3 inhibitor-treated cells. To evaluate antitumor activity, we administered a betacellulin (BTC)-neutralizing antibody alone and in combination with STAT3 inhibition. BTC is an EGFR ligand. We therefore treated mice with orthotopic xenografts using the third-generation EGFR inhibitor osimertinib, with or without STAT3 knockdown.

Results: We demonstrate that both small-molecule inhibitors and knockdown of STAT3 led to expression and secretion of the EGFR ligand BTC, resulting in activation of EGFR and subsequent downstream phosphorylation of nuclear factor-kappaB (NF-κB). Neutralizing antibody against BTC abrogated activation of both EGFR and NF-κB in response to inhibition of STAT3; with combinatorial blockade of STAT3 and BTC inducing apoptosis in GBM cells. Blocking EGFR and STAT3 together inhibited tumor growth, improving survival in mice bearing orthotopic GBM PDXs in vivo.

Conclusion: These data reveal a feedback loop among STAT3, EGFR, and NF-κB that mediates primary resistance to STAT3 blockade and suggest strategies for therapeutic intervention.
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http://dx.doi.org/10.1093/neuonc/noz206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7158663PMC
April 2020

p38γ MAPK contributes to left ventricular remodeling after pathologic stress and disinhibits calpain through phosphorylation of calpastatin.

FASEB J 2019 12 22;33(12):13131-13144. Epub 2019 Oct 22.

School of Cardiovascular Medicine and Science, British Heart Foundation (BHF) Centre, King's College London, London, United Kingdom.

Despite the high and preferential expression of p38γ MAPK in the myocardium, little is known about its function in the heart. The aim of the current study was to elucidate the physiologic and biochemical roles of p38γ in the heart. Expression and subcellular localization of p38 isoforms was determined in mouse hearts. Comparisons of the cardiac function and structure of wild-type and p38γ knockout (KO) mice at baseline and after abdominal aortic banding demonstrated that KO mice developed less ventricular hypertrophy and that contractile function is better preserved. To identify potential substrates of p38γ, we generated an analog-sensitive mutant to affinity tag endogenous myocardial proteins. Among other proteins, this technique identified calpastatin as a direct p38γ substrate. Moreover, phosphorylation of calpastatin by p38γ impaired its ability to inhibit the protease, calpain. We have identified p38γ as an important determinant of the progression of pathologic cardiac hypertrophy after aortic banding in mice. In addition, we have identified calpastatin, among other substrates, as a novel direct target of p38γ that may contribute to the protection observed in p38γKO mice.-Loonat, A. A., Martin, E. D., Sarafraz-Shekary, N., Tilgner, K., Hertz, N. T., Levin, R., Shokat, K. M., Burlingame, A. L., Arabacilar, P., Uddin, S., Thomas, M., Marber, M. S., Clark, J. E. p38γ MAPK contributes to left ventricular remodeling after pathologic stress and disinhibits calpain through phosphorylation of calpastatin.
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http://dx.doi.org/10.1096/fj.201701545RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894093PMC
December 2019

Bifunctional Small-Molecule Ligands of K-Ras Induce Its Association with Immunophilin Proteins.

Angew Chem Int Ed Engl 2019 11 26;58(45):16314-16319. Epub 2019 Sep 26.

Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, 600 16th Street, San Francisco, CA, 94143, USA.

Here we report the design, synthesis, and characterization of bifunctional chemical ligands that induce the association of Ras with ubiquitously expressed immunophilin proteins such as FKBP12 and cyclophilin A. We show this approach is applicable to two distinct Ras ligand scaffolds, and that both the identity of the immunophilin ligand and the linker chemistry affect compound efficacy in biochemical and cellular contexts. These ligands bind to Ras in an immunophilin-dependent fashion and mediate the formation of tripartite complexes of Ras, immunophilin, and the ligand. The recruitment of cyclophilin A to GTP-bound Ras blocks its interaction with B-Raf in biochemical assays. Our study demonstrates the feasibility of ligand-induced association of Ras with intracellular proteins and suggests it as a promising therapeutic strategy for Ras-driven cancers.
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http://dx.doi.org/10.1002/anie.201910124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980260PMC
November 2019

A Bounty of New Challenging Targets in Oncology for Chemical Discovery.

Biochemistry 2019 08 25;58(31):3328-3330. Epub 2019 Jul 25.

Department of Cellular and Molecular Pharmacology , University of California, San Francisco , San Francisco , California 94158 , United States.

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http://dx.doi.org/10.1021/acs.biochem.9b00570DOI Listing
August 2019

KRAS inhibition produces a driver-limited state revealing collateral dependencies.

Sci Signal 2019 05 28;12(583). Epub 2019 May 28.

Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA.

Inhibitors targeting KRAS, a mutant form of the guanosine triphosphatase (GTPase) KRAS, are a promising new class of oncogene-specific therapeutics for the treatment of tumors driven by the mutant protein. These inhibitors react with the mutant cysteine residue by binding covalently to the switch-II pocket (S-IIP) that is present only in the inactive guanosine diphosphate (GDP)-bound form of KRAS, sparing the wild-type protein. We used a genome-scale CRISPR interference (CRISPRi) functional genomics platform to systematically identify genetic interactions with a KRAS inhibitor in cellular models of KRAS mutant lung and pancreatic cancer. Our data revealed genes that were selectively essential in this oncogenic driver-limited cell state, meaning that their loss enhanced cellular susceptibility to direct KRAS inhibition. We termed such genes "collateral dependencies" (CDs) and identified two classes of combination therapies targeting these CDs that increased KRAS target engagement or blocked residual survival pathways in cells and in vivo. From our findings, we propose a framework for assessing genetic dependencies induced by oncogene inhibition.
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http://dx.doi.org/10.1126/scisignal.aaw9450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6871662PMC
May 2019

Phosphoregulation of the oncogenic protein regulator of cytokinesis 1 (PRC1) by the atypical CDK16/CCNY complex.

Exp Mol Med 2019 04 16;51(4):1-17. Epub 2019 Apr 16.

Faculty of Medicine and Health Sciences, International University of Catalonia, 08195, Sant Cugat del Vallès, Barcelona, Spain.

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that forms an active complex with cyclin Y (CCNY). Although both proteins have been recently implicated in cancer pathogenesis, it is still unclear how the CDK16/CCNY complex exerts its biological activity. To understand the CDK16/CCNY network, we used complementary proteomic approaches to identify potential substrates of this complex. We identified several candidates implicating the CDK16/CCNY complex in cytoskeletal dynamics, and we focused on the microtubule-associated protein regulator of cytokinesis (PRC1), an essential protein for cell division that organizes antiparallel microtubules and whose deregulation may drive genomic instability in cancer. Using analog-sensitive (AS) CDK16 generated by CRISPR-Cas9 mutagenesis in 293T cells, we found that specific inhibition of CDK16 induces PRC1 dephosphorylation at Thr481 and delocalization to the nucleus during interphase. The observation that CDK16 inhibition and PRC1 downregulation exhibit epistatic effects on cell viability confirms that these proteins can act through a single pathway. In conclusion, we identified PRC1 as the first substrate of the CDK16/CCNY complex and demonstrated that the proliferative function of CDK16 is mediated by PRC1 phosphorylation. As CDK16 is emerging as a critical node in cancer, our study reveals novel potential therapeutic targets.
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http://dx.doi.org/10.1038/s12276-019-0242-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467995PMC
April 2019

A Legionella pneumophila Kinase Phosphorylates the Hsp70 Chaperone Family to Inhibit Eukaryotic Protein Synthesis.

Cell Host Microbe 2019 03 28;25(3):454-462.e6. Epub 2019 Feb 28.

Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; George Williams Hooper Foundation, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Legionella pneumophila (L.p.), the microbe responsible for Legionnaires' disease, secretes ∼300 bacterial proteins into the host cell cytosol. A subset of these proteins affects a wide range of post-translational modifications (PTMs) to disrupt host cellular pathways. L.p. has 5 conserved eukaryotic-like Ser/Thr effector kinases, LegK1-4 and LegK7, which are translocated during infection. Using a chemical genetic screen, we identified the Hsp70 chaperone family as a direct host target of LegK4. Phosphorylation of Hsp70s at T495 in the substrate-binding domain disrupted Hsp70's ATPase activity and greatly inhibited its protein folding capacity. Phosphorylation of cytosolic Hsp70 by LegK4 resulted in global translation inhibition and an increase in the amount of Hsp70 on highly translating polysomes. LegK4's ability to inhibit host translation via a single PTM uncovers a role for Hsp70 in protein synthesis and directly links it to the cellular translational machinery.
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http://dx.doi.org/10.1016/j.chom.2019.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529236PMC
March 2019

Chronic TGF-β exposure drives stabilized EMT, tumor stemness, and cancer drug resistance with vulnerability to bitopic mTOR inhibition.

Sci Signal 2019 02 26;12(570). Epub 2019 Feb 26.

Department of Cell and Tissue Biology, University of California at San Francisco, San Francisco, CA 94143, USA.

Tumors comprise cancer stem cells (CSCs) and their heterogeneous progeny within a stromal microenvironment. In response to transforming growth factor-β (TGF-β), epithelial and carcinoma cells undergo a partial or complete epithelial-mesenchymal transition (EMT), which contributes to cancer progression. This process is seen as reversible because cells revert to an epithelial phenotype upon TGF-β removal. However, we found that prolonged TGF-β exposure, mimicking the state of in vivo carcinomas, promotes stable EMT in mammary epithelial and carcinoma cells, in contrast to the reversible EMT induced by a shorter exposure. The stabilized EMT was accompanied by stably enhanced stem cell generation and anticancer drug resistance. Furthermore, prolonged TGF-β exposure enhanced mammalian target of rapamycin (mTOR) signaling. A bitopic mTOR inhibitor repressed CSC generation, anchorage independence, cell survival, and chemoresistance and efficiently inhibited tumorigenesis in mice. These results reveal a role for mTOR in the stabilization of stemness and drug resistance of breast cancer cells and position mTOR inhibition as a treatment strategy to target CSCs.
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http://dx.doi.org/10.1126/scisignal.aau8544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746178PMC
February 2019

Chemically reprogramming the phospho-transfer reaction to crosslink protein kinases to their substrates.

Protein Sci 2019 03 31;28(3):654-662. Epub 2019 Jan 31.

Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California.

The proteomic mapping of enzyme-substrate interactions is challenged by their transient nature. A method to capture interacting protein kinases in complexes with a single substrate of interest would provide a new tool for mapping kinase signaling networks. Here, we describe a nucleotide-based substrate analog capable of reprogramming the wild-type phosphoryl-transfer reaction to produce a kinase-acrylamide-based thioether crosslink to mutant substrates with a cysteine nucleophile substituted at the native phosphorylation site. A previously reported ATP-based methacrylate crosslinker (ATP-MA) was capable of mediating kinase crosslinking to short peptides but not protein substrates. Exploration of structural variants of ATP-MA to enable crosslinking of protein substrates to kinases led to the discovery that an ADP-based methacrylate (ADP-MA) crosslinker was superior to the ATP scaffold at crosslinking in vitro. The improved efficiency of ADP-MA over ATP-MA is due to reduced inhibition of the second step of the kinase-substrate crosslinking reaction by the product of the first step of the reaction. The new probe, ADP-MA, demonstrated enhanced in vitro crosslinking between the Src tyrosine kinase and its substrate Cortactin in a phosphorylation site-specific manner. The kinase-substrate crosslinking reaction can be carried out in a complex mammalian cell lysate setting, although the low abundance of endogenous kinases remains a significant challenge for efficient capture.
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http://dx.doi.org/10.1002/pro.3570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371225PMC
March 2019

A Patient-derived Xenograft Model of Pancreatic Neuroendocrine Tumors Identifies Sapanisertib as a Possible New Treatment for Everolimus-resistant Tumors.

Mol Cancer Ther 2018 12 25;17(12):2702-2709. Epub 2018 Sep 25.

Helen Diller Family HDF Comprehensive Cancer Center, University of California, San Francisco, California.

Patients with pancreatic neuroendocrine tumors (PNET) commonly develop advanced disease and require systemic therapy. However, treatment options remain limited, in part, because experimental models that reliably emulate PNET disease are lacking. We therefore developed a patient-derived xenograft model of PNET (PDX-PNET), which we then used to evaluate two mTOR inhibitor drugs: FDA-approved everolimus and the investigational new drug sapanisertib. PDX-PNETs maintained a PNET morphology and PNET-specific gene expression signature with serial passage. PDX-PNETs also harbored mutations in genes previously associated with PNETs (such as and ), displayed activation of the mTOR pathway, and could be detected by Gallium-68 DOTATATE PET-CT. Treatment of PDX-PNETs with either everolimus or sapanisertib strongly inhibited growth. As seen in patients, some PDX-PNETs developed resistance to everolimus. However, sapanisertib, a more potent inhibitor of the mTOR pathway, caused tumor shrinkage in most everolimus-resistant tumors. Our PDX-PNET model is the first available, validated PDX model for PNET, and preclinical data from the use of this model suggest that sapanisertib may be an effective new treatment option for patients with PNET or everolimus-resistant PNET.
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http://dx.doi.org/10.1158/1535-7163.MCT-17-1204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279485PMC
December 2018

Type II Kinase Inhibitors Targeting Cys-Gatekeeper Kinases Display Orthogonality with Wild Type and Ala/Gly-Gatekeeper Kinases.

ACS Chem Biol 2018 10 9;13(10):2956-2965. Epub 2018 Oct 9.

School of Molecular and Cellular Biology, Faculty of Biological Sciences , University of Leeds , Leeds LS2 9JT , U.K.

Analogue-sensitive (AS) kinases contain large to small mutations in the gatekeeper position rendering them susceptible to inhibition with bulky analogues of pyrazolopyrimidine-based Src kinase inhibitors (e.g., PP1). This "bump-hole" method has been utilized for at least 85 of ∼520 kinases, but many kinases are intolerant to this approach. To expand the scope of AS kinase technology, we designed type II kinase inhibitors, ASDO2/6 (analogue-sensitive "DFG-out" kinase inhibitors 2 and 6), that target the "DFG-out" conformation of Cys-gatekeeper kinases with submicromolar potency. We validated this system in vitro against Greatwall kinase (GWL), Aurora-A kinase, and cyclin-dependent kinase-1 and in cells using M110C-GWL-expressing mouse embryonic fibroblasts. These Cys-gatekeeper kinases were sensitive to ASDO2/6 inhibition but not AS kinase inhibitor 3MB-PP1 and vice versa. These compounds, with AS kinase inhibitors, have the potential to inhibit multiple AS kinases independently with applications in systems level and translational kinase research as well as the rational design of type II kinase inhibitors targeting endogenous kinases.
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http://dx.doi.org/10.1021/acschembio.8b00592DOI Listing
October 2018

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9.

J Vis Exp 2018 08 28(138). Epub 2018 Aug 28.

Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California San Francisco.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.
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http://dx.doi.org/10.3791/58265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231911PMC
August 2018

Chemical genetic inhibition of DEAD-box proteins using covalent complementarity.

Nucleic Acids Res 2018 09;46(17):8689-8699

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA.

DEAD-box proteins are an essential class of enzymes involved in all stages of RNA metabolism. The study of DEAD-box proteins is challenging in a native setting since they are structurally similar, often essential and display dosage sensitivity. Pharmacological inhibition would be an ideal tool to probe the function of these enzymes. In this work, we describe a chemical genetic strategy for the specific inactivation of individual DEAD-box proteins with small molecule inhibitors using covalent complementarity. We identify a residue of low conservation within the P-loop of the nucleotide-binding site of DEAD-box proteins and show that it can be mutated to cysteine without a substantial loss of enzyme function to generate electrophile-sensitive mutants. We then present a series of small molecules that rapidly and specifically bind and inhibit electrophile-sensitive DEAD-box proteins with high selectivity over the wild-type enzyme. Thus, this approach can be used to systematically generate small molecule-sensitive alleles of DEAD-box proteins, allowing for pharmacological inhibition and functional characterization of members of this enzyme family.
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http://dx.doi.org/10.1093/nar/gky706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158709PMC
September 2018

Kinome rewiring reveals AURKA limits PI3K-pathway inhibitor efficacy in breast cancer.

Nat Chem Biol 2018 08 25;14(8):768-777. Epub 2018 Jun 25.

Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, USA.

Dysregulation of the PI3K-AKT-mTOR signaling network is a prominent feature of breast cancers. However, clinical responses to drugs targeting this pathway have been modest, possibly because of dynamic changes in cellular signaling that drive resistance and limit drug efficacy. Using a quantitative chemoproteomics approach, we mapped kinome dynamics in response to inhibitors of this pathway and identified signaling changes that correlate with drug sensitivity. Maintenance of AURKA after drug treatment was associated with resistance in breast cancer models. Incomplete inhibition of AURKA was a common source of therapy failure, and combinations of PI3K, AKT or mTOR inhibitors with the AURKA inhibitor MLN8237 were highly synergistic and durably suppressed mTOR signaling, resulting in apoptosis and tumor regression in vivo. This signaling map identifies survival factors whose presence limits the efficacy of targeted therapies and reveals new drug combinations that may unlock the full potential of PI3K-AKT-mTOR pathway inhibitors in breast cancer.
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http://dx.doi.org/10.1038/s41589-018-0081-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6051919PMC
August 2018

Comprehensive analysis of T cell leukemia signals reveals heterogeneity in the PI3 kinase-Akt pathway and limitations of PI3 kinase inhibitors as monotherapy.

PLoS One 2018 25;13(5):e0193849. Epub 2018 May 25.

Department of Anatomy, University of California San Francisco, San Francisco, California, United States of America.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer. Poly-chemotherapy with cytotoxic and genotoxic drugs causes substantial toxicity and more specific therapies targeting the underlying molecular lesions are highly desired. Perturbed Ras signaling is prevalent in T-ALL and occurs via oncogenic RAS mutations or through overexpression of the Ras activator RasGRP1 in ~65% of T-ALL patients. Effective small molecule inhibitors for either target do not currently exist. Genetic and biochemical evidence link phosphoinositide 3-kinase (PI3K) signals to T-ALL, PI3Ks are activated by Ras-dependent and Ras-independent mechanisms, and potent PI3K inhibitors exist. Here we performed comprehensive analyses of PI3K-Akt signaling in T-ALL with a focus on class I PI3K. We developed a multiplex, multiparameter flow cytometry platform with pan- and isoform-specific PI3K inhibitors. We find that pan-PI3K and PI3K γ-specific inhibitors effectively block basal and cytokine-induced PI3K-Akt signals. Despite such inhibition, GDC0941 (pan-PI3K) or AS-605240 (PI3Kγ-specific) as single agents did not efficiently induce death in T-ALL cell lines. Combination of GDC0941 with AS-605240, maximally targeting all p110 isoforms, exhibited potent synergistic activity for clonal T-ALL lines in vitro, which motivated us to perform preclinical trials in mice. In contrast to clonal T-ALL lines, we used a T-ALL cancer model that recapitulates the multi-step pathogenesis and inter- and intra-tumoral genetic heterogeneity, a hallmark of advanced human cancers. We found that the combination of GDC0941 with AS-605240 fails in such trials. Our results reveal that PI3K inhibitors are a promising avenue for molecular therapy in T-ALL, but predict the requirement for methods that can resolve biochemical signals in heterogeneous cell populations so that combination therapy can be designed in a rational manner.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193849PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969748PMC
July 2018

Disease-Causing Mutations in the G Protein Gαs Subvert the Roles of GDP and GTP.

Cell 2018 05 5;173(5):1254-1264.e11. Epub 2018 Apr 5.

Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California-San Francisco, San Francisco, CA 94158, USA; Department of Chemistry, University of California-Berkeley, Berkeley, CA 94720, USA. Electronic address:

The single most frequent cancer-causing mutation across all heterotrimeric G proteins is R201C in Gαs. The current model explaining the gain-of-function activity of the R201 mutations is through the loss of GTPase activity and resulting inability to switch off to the GDP state. Here, we find that the R201C mutation can bypass the need for GTP binding by directly activating GDP-bound Gαs through stabilization of an intramolecular hydrogen bond network. Having found that a gain-of-function mutation can convert GDP into an activator, we postulated that a reciprocal mutation might disrupt the normal role of GTP. Indeed, we found R228C, a loss-of-function mutation in Gαs that causes pseudohypoparathyroidism type 1a (PHP-Ia), compromised the adenylyl cyclase-activating activity of Gαs bound to a non-hydrolyzable GTP analog. These findings show that disease-causing mutations in Gαs can subvert the canonical roles of GDP and GTP, providing new insights into the regulation mechanism of G proteins.
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http://dx.doi.org/10.1016/j.cell.2018.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959768PMC
May 2018

Novel K-Ras G12C Switch-II Covalent Binders Destabilize Ras and Accelerate Nucleotide Exchange.

J Chem Inf Model 2018 02 31;58(2):464-471. Epub 2018 Jan 31.

Department of Organic Chemistry, The Weizmann Institute of Science , Rehovot, 7610001, Israel.

The success of targeted covalent inhibitors in the global pharmaceutical industry has led to a resurgence of covalent drug discovery. However, covalent inhibitor design for flexible binding sites remains a difficult task due to a lack of methodological development. Here, we compared covalent docking to empirical electrophile screening against the highly dynamic target K-Ras. While the overall hit rate of both methods was comparable, we were able to rapidly progress a docking hit to a potent irreversible covalent binder that modifies the inactive, GDP-bound state of K-Ras. Hydrogen-deuterium exchange mass spectrometry was used to probe the protein dynamics of compound binding to the switch-II pocket and subsequent destabilization of the nucleotide-binding region. SOS-mediated nucleotide exchange assays showed that, contrary to prior switch-II pocket inhibitors, these new compounds appear to accelerate nucleotide exchange. This study highlights the efficiency of covalent docking as a tool for the discovery of chemically novel hits against challenging targets.
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http://dx.doi.org/10.1021/acs.jcim.7b00399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6179444PMC
February 2018
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