Publications by authors named "Joshua Pan"

11 Publications

  • Page 1 of 1

Synthetic Lethal Interaction of SHOC2 Depletion with MEK Inhibition in RAS-Driven Cancers.

Cell Rep 2019 10;29(1):118-134.e8

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA; Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 75 Francis Street, Boston, MA 02115, MA. Electronic address:

The mitogen-activated protein kinase (MAPK) pathway is a critical effector of oncogenic RAS signaling, and MAPK pathway inhibition may be an effective combination treatment strategy. We performed genome-scale loss-of-function CRISPR-Cas9 screens in the presence of a MEK1/2 inhibitor (MEKi) in KRAS-mutant pancreatic and lung cancer cell lines and identified genes that cooperate with MEK inhibition. While we observed heterogeneity in genetic modifiers of MEKi sensitivity across cell lines, several recurrent classes of synthetic lethal vulnerabilities emerged at the pathway level. Multiple members of receptor tyrosine kinase (RTK)-RAS-MAPK pathways scored as sensitizers to MEKi. In particular, we demonstrate that knockout, suppression, or degradation of SHOC2, a positive regulator of MAPK signaling, specifically cooperated with MEK inhibition to impair proliferation in RAS-driven cancer cells. The depletion of SHOC2 disrupted survival pathways triggered by feedback RTK signaling in response to MEK inhibition. Thus, these findings nominate SHOC2 as a potential target for combination therapy.
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http://dx.doi.org/10.1016/j.celrep.2019.08.090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918830PMC
October 2019

"Hacking" Our Way across Interdisciplinary Boundaries.

Cell Syst 2019 05;8(5):361-362

Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, USA. Electronic address:

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http://dx.doi.org/10.1016/j.cels.2019.04.006DOI Listing
May 2019

The ATPase module of mammalian SWI/SNF family complexes mediates subcomplex identity and catalytic activity-independent genomic targeting.

Nat Genet 2019 04 11;51(4):618-626. Epub 2019 Mar 11.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

Perturbations to mammalian switch/sucrose non-fermentable (mSWI/SNF) chromatin remodeling complexes have been widely implicated as driving events in cancer. One such perturbation is the dual loss of the SMARCA4 and SMARCA2 ATPase subunits in small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), SMARCA4-deficient thoracic sarcomas and dedifferentiated endometrial carcinomas. However, the consequences of dual ATPase subunit loss on mSWI/SNF complex subunit composition, chromatin targeting, DNA accessibility and gene expression remain unknown. Here we identify an ATPase module of subunits that is required for functional specification of the Brahma-related gene-associated factor (BAF) and polybromo-associated BAF (PBAF) mSWI/SNF family subcomplexes. Using SMARCA4/2 ATPase mutant variants, we define the catalytic activity-dependent and catalytic activity-independent contributions of the ATPase module to the targeting of BAF and PBAF complexes on chromatin genome-wide. Finally, by linking distinct mSWI/SNF complex target sites to tumor-suppressive gene expression programs, we clarify the transcriptional consequences of SMARCA4/2 dual loss in SCCOHT.
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http://dx.doi.org/10.1038/s41588-019-0363-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6755913PMC
April 2019

Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cell 2018 11 25;175(5):1244-1258.e26. Epub 2018 Oct 25.

Fels Institute for Cancer Research, Lewis Katz School of Medicine at Temple University, Philadelphia, PA 19140, USA. Electronic address:

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
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http://dx.doi.org/10.1016/j.cell.2018.09.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6247954PMC
November 2018

A non-canonical SWI/SNF complex is a synthetic lethal target in cancers driven by BAF complex perturbation.

Nat Cell Biol 2018 12 5;20(12):1410-1420. Epub 2018 Nov 5.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA.

Mammalian SWI/SNF chromatin remodelling complexes exist in three distinct, final-form assemblies: canonical BAF (cBAF), PBAF and a newly characterized non-canonical complex (ncBAF). However, their complex-specific targeting on chromatin, functions and roles in disease remain largely undefined. Here, we comprehensively mapped complex assemblies on chromatin and found that ncBAF complexes uniquely localize to CTCF sites and promoters. We identified ncBAF subunits as synthetic lethal targets specific to synovial sarcoma and malignant rhabdoid tumours, which both exhibit cBAF complex (SMARCB1 subunit) perturbation. Chemical and biological depletion of the ncBAF subunit, BRD9, rapidly attenuates synovial sarcoma and malignant rhabdoid tumour cell proliferation. Importantly, in cBAF-perturbed cancers, ncBAF complexes maintain gene expression at retained CTCF-promoter sites and function in a manner distinct from fusion oncoprotein-bound complexes. Together, these findings unmask the unique targeting and functional roles of ncBAF complexes and present new cancer-specific therapeutic targets.
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http://dx.doi.org/10.1038/s41556-018-0221-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698386PMC
December 2018

Modular Organization and Assembly of SWI/SNF Family Chromatin Remodeling Complexes.

Cell 2018 11 18;175(5):1272-1288.e20. Epub 2018 Oct 18.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02215, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.
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http://dx.doi.org/10.1016/j.cell.2018.09.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791824PMC
November 2018

Binding of TMPRSS2-ERG to BAF Chromatin Remodeling Complexes Mediates Prostate Oncogenesis.

Mol Cell 2018 08 2;71(4):554-566.e7. Epub 2018 Aug 2.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA; Broad Institute of Harvard and MIT, Cambridge, MA, USA. Electronic address:

Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.
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http://dx.doi.org/10.1016/j.molcel.2018.06.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140332PMC
August 2018

Interrogation of Mammalian Protein Complex Structure, Function, and Membership Using Genome-Scale Fitness Screens.

Cell Syst 2018 05 16;6(5):555-568.e7. Epub 2018 May 16.

Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; Broad Institute of Harvard and MIT, 415 Main Street, Cambridge, MA 02142, USA; Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Protein complexes are assemblies of subunits that have co-evolved to execute one or many coordinated functions in the cellular environment. Functional annotation of mammalian protein complexes is critical to understanding biological processes, as well as disease mechanisms. Here, we used genetic co-essentiality derived from genome-scale RNAi- and CRISPR-Cas9-based fitness screens performed across hundreds of human cancer cell lines to assign measures of functional similarity. From these measures, we systematically built and characterized functional similarity networks that recapitulate known structural and functional features of well-studied protein complexes and resolve novel functional modules within complexes lacking structural resolution, such as the mammalian SWI/SNF complex. Finally, by integrating functional networks with large protein-protein interaction networks, we discovered novel protein complexes involving recently evolved genes of unknown function. Taken together, these findings demonstrate the utility of genetic perturbation screens alone, and in combination with large-scale biophysical data, to enhance our understanding of mammalian protein complexes in normal and disease states.
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http://dx.doi.org/10.1016/j.cels.2018.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152908PMC
May 2018

IL-1β Inhibits Connexin 43 and Disrupts Decidualization of Human Endometrial Stromal Cells Through ERK1/2 and p38 MAP Kinase.

Endocrinology 2017 12;158(12):4270-4285

Department of Obstetrics and Gynecology, Wake Forest School of Medicine.

Inflammation can interfere with endometrial receptivity. We examined how interleukin 1β (IL-1β) affects expression of the uterine gap junction protein connexin 43 (Cx43), which is known to be critical for embryonic implantation. We used an in vitro model of human endometrial stromal cells (ESCs), Western blotting, and a combination of validated, selective kinase inhibitors to evaluate five canonical IL-1β signaling pathways. Cx43 and two other markers of ESC differentiation (prolactin and VEGF) were inhibited predominantly via IL-1β-activated ERK1/2 and p38 MAP kinase cascades. The findings were corroborated using small interfering RNA to silence critical genes in either pathway. By contrast, upregulation of endogenous pro-IL-1α and pro-IL-1β following recombinant IL-1β treatment was mediated via the Jun N-terminal kinase pathway. The clinicopharmacological significance of our findings is that multiple signaling cascades may need to be neutralized to reverse deleterious effects of IL-1β on human endometrial function.
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http://dx.doi.org/10.1210/en.2017-00495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711380PMC
December 2017

Combined Spinal Epidural Technique for Labor Analgesia Does Not Delay Recognition of Epidural Catheter Failures: A Single-center Retrospective Cohort Survival Analysis.

Anesthesiology 2016 09;125(3):516-24

From the Section of Obstetric Anesthesia, Department of Anesthesiology (J.M.B., J.C.P., V.H.R., L.C.H., P.H.P.) and Department of Biostatistical Sciences (G.B.R.), Wake Forest School of Medicine, Winston-Salem, North Carolina.

Background: It is unclear whether recognition of epidural catheter failures is delayed with combined spinal epidural technique (CSE) compared to traditional epidural technique (EPID) when used for labor analgesia. The authors hypothesized that recognition of failed catheters is not delayed by CSE.

Methods: Anesthetic, obstetric, and quality assurance records from 2,395 labor neuraxial procedures (1,440 CSE and 955 EPID) performed at Forsyth Medical Center (Winston-Salem, North Carolina) between June 30 and December 31, 2012, were retrospectively analyzed. The primary outcome was catheter survival (failure-free) time during labor analgesia. A proportional hazards model with the counting method was used to assess relationships between the techniques and survival (failure-free) time of catheters, while controlling for subjects' body mass index and providers' level of training in the final best-fit multivariable regression model.

Results: Cumulative incidence of epidural catheter failures was 6.6% for CSE and 11.6% for EPID (P = 0.001). In the multivariable regression model, catheters placed with CSE versus epidural were less likely to fail (hazard ratio, 0.58; 95% CI, 0.43 to 0.79; P = 0.0002) for labor analgesia. Among the catheters that failed, there was no overall difference in failure time course between the techniques (hazard ratio, 1.17; 95% CI, 0.89 to 1.54; P = 0.26) even though more failed catheters with CSE (48.4%) than with EPID (30.6%) were recognized within the first 30 min of placement (P = 0.009).

Conclusions: In this cohort, CSE has a significantly lower risk of overall epidural catheter failures than EPID and does not delay recognition of epidural catheter failures. Choice of CSE versus EPID should be based on overall risk of failure, efficacy, and side effects.
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http://dx.doi.org/10.1097/ALN.0000000000001222DOI Listing
September 2016

CRISPR/Cas9-Directed Genome Editing of Cultured Cells.

Curr Protoc Mol Biol 2014 Jul 1;107:31.1.1-17. Epub 2014 Jul 1.

Department of Genetics, Harvard Medical School, Boston, Massachusetts.

Human genome engineering has been transformed by the introduction of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system found in most bacteria and archaea. Type II CRISPR/Cas systems have been engineered to induce RNA-guided genome editing in human cells, where small RNAs function together with Cas9 nucleases for sequence-specific cleavage of target sequences. Here we describe the protocol for Cas9-mediated human genome engineering, including construct building and transfection methods necessary for delivering Cas9 and guide RNA (gRNA) into human-induced pluripotent stem cells (hiPSCs) and HEK293 cells. Following genome editing, we also describe methods to assess genome editing efficiency using next-generation sequencing and isolate monoclonal hiPSCs with the desired modifications for downstream applications.
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http://dx.doi.org/10.1002/0471142727.mb3101s107DOI Listing
July 2014