Publications by authors named "David E Root"

154 Publications

SMAD4 represses FOSL1 expression and pancreatic cancer metastatic colonization.

Cell Rep 2021 Jul;36(4):109443

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Metastasis is a complex and poorly understood process. In pancreatic cancer, loss of the transforming growth factor (TGF)-β/BMP effector SMAD4 is correlated with changes in altered histopathological transitions, metastatic disease, and poor prognosis. In this study, we use isogenic cancer cell lines to identify SMAD4 regulated genes that contribute to the development of metastatic colonization. We perform an in vivo screen identifying FOSL1 as both a SMAD4 target and sufficient to drive colonization to the lung. The targeting of these genes early in treatment may provide a therapeutic benefit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2021.109443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8350598PMC
July 2021

Acquired Resistance to KRAS Inhibition in Cancer.

N Engl J Med 2021 06;384(25):2382-2393

From Dana-Farber Cancer Institute (M.M.A., S.L., J.D., J.O.J., K.E.L., H.F., K.M.H., B.M.W., P.A.J., A.J.A.), Massachusetts General Hospital (R.S.H., Y.P.H.), and Brigham and Women's Hospital (L.M.S., A.J.A.), Boston, and Broad Institute of MIT and Harvard (S.L., X.Y., N.S.P., D.E.R., K.M.H., A.J.A.) and Foundation Medicine (J.L., A.B.S.), Cambridge - all in Massachusetts; Henry Ford Cancer Institute, Detroit (I.I.R.); Memorial Sloan Kettering Cancer Center, New York (K.C.A., G.J.R., P.L.); Chao Family Comprehensive Cancer Center, University of California, Irvine, School of Medicine, Orange (V.W.Z., S.S.Z., S.-H.I.O.), Boundless Bio, La Jolla (J.W., J.C.), and Mirati Therapeutics, San Diego (L.D.E., L.W., J.D.L., P.O., J.G.C.) - all in California; Sarah Cannon Research Institute, Tennessee Oncology/OneOncology, Nashville (M.L.J.); the University of Colorado, Aurora (T.P.); and Resolution Bioscience, Kirkland, WA (L.P.L., K.G., M.L.).

Background: Clinical trials of the KRAS inhibitors adagrasib and sotorasib have shown promising activity in cancers harboring KRAS glycine-to-cysteine amino acid substitutions at codon 12 (KRAS). The mechanisms of acquired resistance to these therapies are currently unknown.

Methods: Among patients with -mutant cancers treated with adagrasib monotherapy, we performed genomic and histologic analyses that compared pretreatment samples with those obtained after the development of resistance. Cell-based experiments were conducted to study mutations that confer resistance to KRAS inhibitors.

Results: A total of 38 patients were included in this study: 27 with non-small-cell lung cancer, 10 with colorectal cancer, and 1 with appendiceal cancer. Putative mechanisms of resistance to adagrasib were detected in 17 patients (45% of the cohort), of whom 7 (18% of the cohort) had multiple coincident mechanisms. Acquired alterations included G12D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, and high-level amplification of the allele. Acquired bypass mechanisms of resistance included amplification; activating mutations in , , , and ; oncogenic fusions involving , , , , and ; and loss-of-function mutations in and . In two of nine patients with lung adenocarcinoma for whom paired tissue-biopsy samples were available, histologic transformation to squamous-cell carcinoma was observed without identification of any other resistance mechanisms. Using an in vitro deep mutational scanning screen, we systematically defined the landscape of mutations that confer resistance to KRAS inhibitors.

Conclusions: Diverse genomic and histologic mechanisms impart resistance to covalent KRAS inhibitors, and new therapeutic strategies are required to delay and overcome this drug resistance in patients with cancer. (Funded by Mirati Therapeutics and others; ClinicalTrials.gov number, NCT03785249.).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1056/NEJMoa2105281DOI Listing
June 2021

Selective Modulation of a Pan-Essential Protein as a Therapeutic Strategy in Cancer.

Cancer Discov 2021 Sep 21;11(9):2282-2299. Epub 2021 Apr 21.

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

Cancer dependency maps, which use CRISPR/Cas9 depletion screens to profile the landscape of genetic dependencies in hundreds of cancer cell lines, have identified context-specific dependencies that could be therapeutically exploited. An ideal therapy is both lethal and precise, but these depletion screens cannot readily distinguish between gene effects that are cytostatic or cytotoxic. Here, we use a diverse panel of functional genomic screening assays to identify as a selective and rapidly lethal relevant genetic dependency in -amplified neuroblastoma. NXT1 heterodimerizes with NXF1, and together they form the principal mRNA nuclear export machinery. We describe a previously unrecognized mechanism of synthetic lethality between NXT1 and its paralog NXT2: their common essential binding partner NXF1 is lost only in the absence of both. We propose a potential therapeutic strategy for tumor-selective elimination of a protein that, if targeted directly, is expected to cause widespread toxicity. SIGNIFICANCE: We provide a framework for identifying new therapeutic targets from functional genomic screens. We nominate NXT1 as a selective lethal target in neuroblastoma and propose a therapeutic approach where the essential protein NXF1 can be selectively eliminated in tumor cells by exploiting the NXT1-NXT2 paralog relationship...
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-20-1213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8419012PMC
September 2021

LKB1/ Is a Tumor Suppressor in the Progression of Myeloproliferative Neoplasms.

Cancer Discov 2021 Jun 12;11(6):1398-1410. Epub 2021 Feb 12.

Northwestern University, Chicago, Illinois.

The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/ led to enhanced self-renewal of murine MPN cells. Deletion of in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells . LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that is a tumor suppressor in the MPNs. SIGNIFICANCE: Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/ leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression..
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-20-1353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8178182PMC
June 2021

Noncanonical open reading frames encode functional proteins essential for cancer cell survival.

Nat Biotechnol 2021 06 28;39(6):697-704. Epub 2021 Jan 28.

Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Although genomic analyses predict many noncanonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442-renamed glycine-rich extracellular protein-1 (GREP1)-encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41587-020-00806-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8195866PMC
June 2021

Synthetic Lethal Interaction between the ESCRT Paralog Enzymes VPS4A and VPS4B in Cancers Harboring Loss of Chromosome 18q or 16q.

Cell Rep 2020 12;33(11):108493

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

Few therapies target the loss of tumor suppressor genes in cancer. We examine CRISPR-SpCas9 and RNA-interference loss-of-function screens to identify new therapeutic targets associated with genomic loss of tumor suppressor genes. The endosomal sorting complexes required for transport (ESCRT) ATPases VPS4A and VPS4B score as strong synthetic lethal dependencies. VPS4A is essential in cancers harboring loss of VPS4B adjacent to SMAD4 on chromosome 18q and VPS4B is required in tumors with co-deletion of VPS4A and CDH1 (E-cadherin) on chromosome 16q. We demonstrate that more than 30% of cancers selectively require VPS4A or VPS4B. VPS4A suppression in VPS4B-deficient cells selectively leads to ESCRT-III filament accumulation, cytokinesis defects, nuclear deformation, G2/M arrest, apoptosis, and potent tumor regression. CRISPR-SpCas9 screening and integrative genomic analysis reveal other ESCRT members, regulators of abscission, and interferon signaling as modifiers of VPS4A dependency. We describe a compendium of synthetic lethal vulnerabilities and nominate VPS4A and VPS4B as high-priority therapeutic targets for cancers with 18q or 16q loss.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.108493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8374858PMC
December 2020

Functional Genomics Identifies Metabolic Vulnerabilities in Pancreatic Cancer.

Cell Metab 2021 01 4;33(1):199-210.e8. Epub 2020 Nov 4.

Department of Radiation Oncology, Perlmutter Cancer Center, New York University Medical Center, New York, NY 10016, USA. Electronic address:

Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer characterized by complex metabolic adaptations that promote survival in a severely hypoxic and nutrient-limited tumor microenvironment (TME). Modeling microenvironmental influences in cell culture has been challenging, and technical limitations have hampered the comprehensive study of tumor-specific metabolism in vivo. To systematically interrogate metabolic vulnerabilities in PDA, we employed parallel CRISPR-Cas9 screens using in vivo and in vitro systems. This work revealed striking overlap of in vivo metabolic dependencies with those in vitro. Moreover, we identified that intercellular nutrient sharing can mask dependencies in pooled screens, highlighting a limitation of this approach to study tumor metabolism. Furthermore, metabolic dependencies were similar between 2D and 3D culture, although 3D culture may better model vulnerabilities that influence certain oncogenic signaling pathways. Lastly, our work demonstrates the power of genetic screening approaches to define in vivo metabolic dependencies and pathways that may have therapeutic utility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cmet.2020.10.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790858PMC
January 2021

Progression signature underlies clonal evolution and dissemination of multiple myeloma.

Blood 2021 Apr;137(17):2360-2372

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

Clonal evolution drives tumor progression, dissemination, and relapse in multiple myeloma (MM), with most patients dying of relapsed disease. This multistage process requires tumor cells to enter the circulation, extravasate, and colonize distant bone marrow (BM) sites. Here, we developed a fluorescent or DNA-barcode clone-tracking system on MM PrEDiCT (progression through evolution and dissemination of clonal tumor cells) xenograft mouse model to study clonal behavior within the BM microenvironment. We showed that only the few clones that successfully adapt to the BM microenvironment can enter the circulation and colonize distant BM sites. RNA sequencing of primary and distant-site MM tumor cells revealed a progression signature sequentially activated along human MM progression and significantly associated with overall survival when evaluated against patient data sets. A total of 28 genes were then computationally predicted to be master regulators (MRs) of MM progression. HMGA1 and PA2G4 were validated in vivo using CRISPR-Cas9 in the PrEDiCT model and were shown to be significantly depleted in distant BM sites, indicating their role in MM progression and dissemination. Loss of HMGA1 and PA2G4 also compromised the proliferation, migration, and adhesion abilities of MM cells in vitro. Overall, our model successfully recapitulates key characteristics of human MM disease progression and identified potential new therapeutic targets for MM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2020005885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085483PMC
April 2021

Acquired FGFR and FGF Alterations Confer Resistance to Estrogen Receptor (ER) Targeted Therapy in ER Metastatic Breast Cancer.

Clin Cancer Res 2020 11 28;26(22):5974-5989. Epub 2020 Jul 28.

Center for Cancer Precision Medicine, Dana-Farber Cancer Institute, Boston, Massachusetts.

Purpose: To identify clinically relevant mechanisms of resistance to ER-directed therapies in ER breast cancer.

Experimental Design: We conducted a genome-scale functional screen spanning 10,135 genes to investigate genes whose overexpression confer resistance to selective estrogen receptor degraders. In parallel, we performed whole-exome sequencing in paired pretreatment and postresistance biopsies from 60 patients with ER metastatic breast cancer who had developed resistance to ER-targeted therapy. Furthermore, we performed experiments to validate resistance genes/pathways and to identify drug combinations to overcome resistance.

Results: Pathway analysis of candidate resistance genes demonstrated that the FGFR, ERBB, insulin receptor, and MAPK pathways represented key modalities of resistance. The FGFR pathway was altered via , or amplifications or mutations in 24 (40%) of the postresistance biopsies. In 12 of the 24 postresistance tumors exhibiting FGFR/FGF alterations, these alterations were acquired or enriched under the selective pressure of ER-directed therapy. experiments in ER breast cancer cells confirmed that FGFR/FGF alterations led to fulvestrant resistance as well as cross-resistance to the CDK4/6 inhibitor palbociclib. RNA sequencing of resistant cell lines demonstrated that FGFR/FGF induced resistance through ER reprogramming and activation of the MAPK pathway. The resistance phenotypes were reversed by FGFR inhibitors, a MEK inhibitor, and/or a SHP2 inhibitor.

Conclusions: Our results suggest that FGFR pathway is a distinct mechanism of acquired resistance to ER-directed therapy that can be overcome by FGFR and/or MAPK pathway inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-19-3958DOI Listing
November 2020

A genome-wide gain-of-function screen identifies CDKN2C as a HBV host factor.

Nat Commun 2020 06 1;11(1):2707. Epub 2020 Jun 1.

Université de Strasbourg, Inserm, Institut de Recherche sur les Maladies Virales et Hépatiques UMR_S1110, F-67000, Strasbourg, France.

Chronic HBV infection is a major cause of liver disease and cancer worldwide. Approaches for cure are lacking, and the knowledge of virus-host interactions is still limited. Here, we perform a genome-wide gain-of-function screen using a poorly permissive hepatoma cell line to uncover host factors enhancing HBV infection. Validation studies in primary human hepatocytes identified CDKN2C as an important host factor for HBV replication. CDKN2C is overexpressed in highly permissive cells and HBV-infected patients. Mechanistic studies show a role for CDKN2C in inducing cell cycle G1 arrest through inhibition of CDK4/6 associated with the upregulation of HBV transcription enhancers. A correlation between CDKN2C expression and disease progression in HBV-infected patients suggests a role in HBV-induced liver disease. Taken together, we identify a previously undiscovered clinically relevant HBV host factor, allowing the development of improved infectious model systems for drug discovery and the study of the HBV life cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-16517-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264273PMC
June 2020

A Genome-scale CRISPR Screen Identifies the ERBB and mTOR Signaling Networks as Key Determinants of Response to PI3K Inhibition in Pancreatic Cancer.

Mol Cancer Ther 2020 07 5;19(7):1423-1435. Epub 2020 May 5.

Division of Cancer Therapeutics, The Institute of Cancer Research, London, United Kingdom.

mutation is a key driver of pancreatic cancer and PI3K pathway activity is an additional requirement for -induced tumorigenesis. Clinical trials of PI3K pathway inhibitors in pancreatic cancer have shown limited responses. Understanding the molecular basis for this lack of efficacy may direct future treatment strategies with emerging PI3K inhibitors. We sought new therapeutic approaches that synergize with PI3K inhibitors through pooled CRISPR modifier genetic screening and a drug combination screen. ERBB family receptor tyrosine kinase signaling and mTOR signaling were key modifiers of sensitivity to alpelisib and pictilisib. Inhibition of the ERBB family or mTOR was synergistic with PI3K inhibition in spheroid, stromal cocultures. Near-complete loss of ribosomal S6 phosphorylation was associated with synergy. Genetic alterations in the ERBB-PI3K signaling axis were associated with decreased survival of patients with pancreatic cancer. Suppression of the PI3K/mTOR axis is potentiated by dual PI3K and ERBB family or mTOR inhibition. Surprisingly, despite the presence of oncogenic KRAS, thought to bestow independence from receptor tyrosine kinase signaling, inhibition of the ERBB family blocks downstream pathway activation and synergizes with PI3K inhibitors. Further exploration of these therapeutic combinations is warranted for the treatment of pancreatic cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-19-1131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611271PMC
July 2020

Aire-dependent genes undergo Clp1-mediated 3'UTR shortening associated with higher transcript stability in the thymus.

Elife 2020 04 29;9. Epub 2020 Apr 29.

Institut Cochin, INSERM U1016, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.

The ability of the immune system to avoid autoimmune disease relies on tolerization of thymocytes to self-antigens whose expression and presentation by thymic medullary epithelial cells (mTECs) is controlled predominantly by Aire at the transcriptional level and possibly regulated at other unrecognized levels. Aire-sensitive gene expression is influenced by several molecular factors, some of which belong to the 3'end processing complex, suggesting they might impact transcript stability and levels through an effect on 3'UTR shortening. We discovered that Aire-sensitive genes display a pronounced preference for short-3'UTR transcript isoforms in mTECs, a feature preceding Aire's expression and correlated with the preferential selection of proximal polyA sites by the 3'end processing complex. Through an RNAi screen and generation of a lentigenic mouse, we found that one factor, Clp1, promotes 3'UTR shortening associated with higher transcript stability and expression of Aire-sensitive genes, revealing a post-transcriptional level of control of Aire-activated expression in mTECs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.52985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205469PMC
April 2020

Agreement between two large pan-cancer CRISPR-Cas9 gene dependency data sets.

Nat Commun 2019 12 20;10(1):5817. Epub 2019 Dec 20.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

Genome-scale CRISPR-Cas9 viability screens performed in cancer cell lines provide a systematic approach to identify cancer dependencies and new therapeutic targets. As multiple large-scale screens become available, a formal assessment of the reproducibility of these experiments becomes necessary. We analyze data from recently published pan-cancer CRISPR-Cas9 screens performed at the Broad and Sanger Institutes. Despite significant differences in experimental protocols and reagents, we find that the screen results are highly concordant across multiple metrics with both common and specific dependencies jointly identified across the two studies. Furthermore, robust biomarkers of gene dependency found in one data set are recovered in the other. Through further analysis and replication experiments at each institute, we show that batch effects are driven principally by two key experimental parameters: the reagent library and the assay length. These results indicate that the Broad and Sanger CRISPR-Cas9 viability screens yield robust and reproducible findings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-13805-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925302PMC
December 2019

CREB5 Promotes Resistance to Androgen-Receptor Antagonists and Androgen Deprivation in Prostate Cancer.

Cell Rep 2019 11;29(8):2355-2370.e6

Dana-Farber Cancer Institute, Boston, MA, USA; Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Androgen-receptor (AR) inhibitors, including enzalutamide, are used for treatment of all metastatic castration-resistant prostate cancers (mCRPCs). However, some patients develop resistance or never respond. We find that the transcription factor CREB5 confers enzalutamide resistance in an open reading frame (ORF) expression screen and in tumor xenografts. CREB5 overexpression is essential for an enzalutamide-resistant patient-derived organoid. In AR-expressing prostate cancer cells, CREB5 interactions enhance AR activity at a subset of promoters and enhancers upon enzalutamide treatment, including MYC and genes involved in the cell cycle. In mCRPC, we found recurrent amplification and overexpression of CREB5. Our observations identify CREB5 as one mechanism that drives resistance to AR antagonists in prostate cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.10.068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886683PMC
November 2019

A Compendium of Genetic Modifiers of Mitochondrial Dysfunction Reveals Intra-organelle Buffering.

Cell 2019 11;179(5):1222-1238.e17

Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114, USA, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA. Electronic address:

Mitochondrial dysfunction is associated with a spectrum of human conditions, ranging from rare, inborn errors of metabolism to the aging process. To identify pathways that modify mitochondrial dysfunction, we performed genome-wide CRISPR screens in the presence of small-molecule mitochondrial inhibitors. We report a compendium of chemical-genetic interactions involving 191 distinct genetic modifiers, including 38 that are synthetic sick/lethal and 63 that are suppressors. Genes involved in glycolysis (PFKP), pentose phosphate pathway (G6PD), and defense against lipid peroxidation (GPX4) scored high as synthetic sick/lethal. A surprisingly large fraction of suppressors are pathway intrinsic and encode mitochondrial proteins. A striking example of such "intra-organelle" buffering is the alleviation of a chemical defect in complex V by simultaneous inhibition of complex I, which benefits cells by rebalancing redox cofactors, increasing reductive carboxylation, and promoting glycolysis. Perhaps paradoxically, certain forms of mitochondrial dysfunction may best be buffered with "second site" inhibitors to the organelle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.10.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053407PMC
November 2019

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.08.090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918830PMC
October 2019

Small-Molecule and CRISPR Screening Converge to Reveal Receptor Tyrosine Kinase Dependencies in Pediatric Rhabdoid Tumors.

Cell Rep 2019 08;28(9):2331-2344.e8

Comprehensive Cancer Center and Department of Oncology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. Electronic address:

Cancer is often seen as a disease of mutations and chromosomal abnormalities. However, some cancers, including pediatric rhabdoid tumors (RTs), lack recurrent alterations targetable by current drugs and need alternative, informed therapeutic options. To nominate potential targets, we performed a high-throughput small-molecule screen complemented by a genome-scale CRISPR-Cas9 gene-knockout screen in a large number of RT and control cell lines. These approaches converged to reveal several receptor tyrosine kinases (RTKs) as therapeutic targets, with RTK inhibition effective in suppressing RT cell growth in vitro and against a xenograft model in vivo. RT cell lines highly express and activate (phosphorylate) different RTKs, creating dependency without mutation or amplification. Downstream of RTK signaling, we identified PTPN11, encoding the pro-growth signaling protein SHP2, as a shared dependency across all RT cell lines. This study demonstrates that large-scale perturbational screening can uncover vulnerabilities in cancers with "quiet" genomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.07.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319190PMC
August 2019

A dominant-negative effect drives selection of missense mutations in myeloid malignancies.

Science 2019 08;365(6453):599-604

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

, which encodes the tumor suppressor p53, is the most frequently mutated gene in human cancer. The selective pressures shaping its mutational spectrum, dominated by missense mutations, are enigmatic, and neomorphic gain-of-function (GOF) activities have been implicated. We used CRISPR-Cas9 to generate isogenic human leukemia cell lines of the most common missense mutations. Functional, DNA-binding, and transcriptional analyses revealed loss of function but no GOF effects. Comprehensive mutational scanning of p53 single-amino acid variants demonstrated that missense variants in the DNA-binding domain exert a dominant-negative effect (DNE). In mice, the DNE of p53 missense variants confers a selective advantage to hematopoietic cells on DNA damage. Analysis of clinical outcomes in patients with acute myeloid leukemia showed no evidence of GOF for missense mutations. Thus, a DNE is the primary unit of selection for missense mutations in myeloid malignancies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aax3649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327437PMC
August 2019

Neuronal differentiation and cell-cycle programs mediate response to BET-bromodomain inhibition in MYC-driven medulloblastoma.

Nat Commun 2019 06 3;10(1):2400. Epub 2019 Jun 3.

Department of Pathology, Brigham and Women's Hospital, Boston, USA.

BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately for resistance, have not been fully defined. Here, using a combination of expression profiling, genome-scale CRISPR/Cas9-mediated loss of function and ORF/cDNA driven rescue screens, and cell-based models of spontaneous resistance, we identify bHLH/homeobox transcription factors and cell-cycle regulators as key genes mediating BETi's response and resistance. Cells that acquire drug tolerance exhibit a more neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they do not terminally differentiate, maintain expression of CCND2, and continue to cycle through S-phase. Moreover, CDK4/CDK6 inhibition delays acquisition of resistance. Therefore, our data provide insights about the mechanisms underlying BETi effects and the appearance of resistance and support the therapeutic use of combined cell-cycle inhibitors with BETi in MYC-amplified medulloblastoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-10307-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546744PMC
June 2019

Gene-centric functional dissection of human genetic variation uncovers regulators of hematopoiesis.

Elife 2019 05 9;8. Epub 2019 May 9.

Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, United States.

Genome-wide association studies (GWAS) have identified thousands of variants associated with human diseases and traits. However, the majority of GWAS-implicated variants are in non-coding regions of the genome and require in depth follow-up to identify target genes and decipher biological mechanisms. Here, rather than focusing on causal variants, we have undertaken a pooled loss-of-function screen in primary hematopoietic cells to interrogate 389 candidate genes contained in 75 loci associated with red blood cell traits. Using this approach, we identify 77 genes at 38 GWAS loci, with most loci harboring 1-2 candidate genes. Importantly, the hit set was strongly enriched for genes validated through orthogonal genetic approaches. Genes identified by this approach are enriched in specific and relevant biological pathways, allowing regulators of human erythropoiesis and modifiers of blood diseases to be defined. More generally, this functional screen provides a paradigm for gene-centric follow up of GWAS for a variety of human diseases and traits.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.44080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534380PMC
May 2019

The landscape of cancer cell line metabolism.

Nat Med 2019 05 8;25(5):850-860. Epub 2019 May 8.

Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Despite considerable efforts to identify cancer metabolic alterations that might unveil druggable vulnerabilities, systematic characterizations of metabolism as it relates to functional genomic features and associated dependencies remain uncommon. To further understand the metabolic diversity of cancer, we profiled 225 metabolites in 928 cell lines from more than 20 cancer types in the Cancer Cell Line Encyclopedia (CCLE) using liquid chromatography-mass spectrometry (LC-MS). This resource enables unbiased association analysis linking the cancer metabolome to genetic alterations, epigenetic features and gene dependencies. Additionally, by screening barcoded cell lines, we demonstrated that aberrant ASNS hypermethylation sensitizes subsets of gastric and hepatic cancers to asparaginase therapy. Finally, our analysis revealed distinct synthesis and secretion patterns of kynurenine, an immune-suppressive metabolite, in model cancer cell lines. Together, these findings and related methodology provide comprehensive resources that will help clarify the landscape of cancer metabolism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-019-0404-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629041PMC
May 2019

WRN helicase is a synthetic lethal target in microsatellite unstable cancers.

Nature 2019 04 10;568(7753):551-556. Epub 2019 Apr 10.

Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1102-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580861PMC
April 2019

Renal medullary carcinomas depend upon loss and are sensitive to proteasome inhibition.

Elife 2019 03 12;8. Epub 2019 Mar 12.

Broad Institute of Harvard and MIT, Cambridge, United States.

Renal medullary carcinoma (RMC) is a rare and deadly kidney cancer in patients of African descent with sickle cell trait. We have developed faithful patient-derived RMC models and using whole-genome sequencing, we identified loss-of-function intronic fusion events in one allele with concurrent loss of the other allele. Biochemical and functional characterization of these models revealed that RMC requires the loss of for survival. Through integration of RNAi and CRISPR-Cas9 loss-of-function genetic screens and a small-molecule screen, we found that the ubiquitin-proteasome system (UPS) was essential in RMC. Inhibition of the UPS caused a G2/M arrest due to constitutive accumulation of cyclin B1. These observations extend across cancers that harbor loss, which also require expression of the E2 ubiquitin-conjugating enzyme, . Our studies identify a synthetic lethal relationship between -deficient cancers and reliance on the UPS which provides the foundation for a mechanism-informed clinical trial with proteasome inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.44161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436895PMC
March 2019

A Functional Landscape of Resistance to MEK1/2 and CDK4/6 Inhibition in NRAS-Mutant Melanoma.

Cancer Res 2019 05 28;79(9):2352-2366. Epub 2019 Feb 28.

Cancer Program, The Broad Institute of M.I.T. and Harvard, Cambridge, Massachusetts.

Combinatorial inhibition of MEK1/2 and CDK4/6 is currently undergoing clinical investigation in NRAS-mutant melanoma. To prospectively map the landscape of resistance to this investigational regimen, we utilized a series of gain- and loss-of-function forward genetic screens to identify modulators of resistance to clinical inhibitors of MEK1/2 and CDK4/6 alone and in combination. First, we identified NRAS-mutant melanoma cell lines that were dependent on NRAS for proliferation and sensitive to MEK1/2 and CDK4/6 combination treatment. We then used a genome-scale ORF overexpression screen and a CRISPR knockout screen to identify modulators of resistance to each inhibitor alone or in combination. These orthogonal screening approaches revealed concordant means of achieving resistance to this therapeutic modality, including tyrosine kinases, RAF, RAS, AKT, and PI3K signaling. Activated KRAS was sufficient to cause resistance to combined MEK/CDK inhibition and to replace genetic depletion of oncogenic NRAS. In summary, our comprehensive functional genetic screening approach revealed modulation of resistance to the inhibition of MEK1/2, CDK4/6, or their combination in NRAS-mutant melanoma. SIGNIFICANCE: These findings reveal that NRAS-mutant melanomas can acquire resistance to genetic ablation of or combination MEK1/2 and CDK4/6 inhibition by upregulating activity of the RTK-RAS-RAF and RTK-PI3K-AKT signaling cascade.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-18-2711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227487PMC
May 2019

Small-molecule targeting of brachyury transcription factor addiction in chordoma.

Nat Med 2019 02 21;25(2):292-300. Epub 2019 Jan 21.

Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Chordoma is a primary bone cancer with no approved therapy. The identification of therapeutic targets in this disease has been challenging due to the infrequent occurrence of clinically actionable somatic mutations in chordoma tumors. Here we describe the discovery of therapeutically targetable chordoma dependencies via genome-scale CRISPR-Cas9 screening and focused small-molecule sensitivity profiling. These systematic approaches reveal that the developmental transcription factor T (brachyury; TBXT) is the top selectively essential gene in chordoma, and that transcriptional cyclin-dependent kinase (CDK) inhibitors targeting CDK7/12/13 and CDK9 potently suppress chordoma cell proliferation. In other cancer types, transcriptional CDK inhibitors have been observed to downregulate highly expressed, enhancer-associated oncogenic transcription factors. In chordoma, we find that T is associated with a 1.5-Mb region containing 'super-enhancers' and is the most highly expressed super-enhancer-associated transcription factor. Notably, transcriptional CDK inhibition leads to preferential and concentration-dependent downregulation of cellular brachyury protein levels in all models tested. In vivo, CDK7/12/13-inhibitor treatment substantially reduces tumor growth. Together, these data demonstrate small-molecule targeting of brachyury transcription factor addiction in chordoma, identify a mechanism of T gene regulation that underlies this therapeutic strategy, and provide a blueprint for applying systematic genetic and chemical screening approaches to discover vulnerabilities in genomically quiet cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-018-0312-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633917PMC
February 2019

Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities.

Nat Commun 2018 12 21;9(1):5416. Epub 2018 Dec 21.

Broad Institute of Harvard and MIT, 75 Ames Street, Cambridge, MA, 02142, USA.

The creation of genome-wide libraries for CRISPR knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa) has enabled the systematic interrogation of gene function. Here, we show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we present genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese), and show in negative selection screens that Dolcetto, with fewer sgRNAs per gene, outperforms existing CRISPRi libraries and achieves comparable performance to CRISPRko in detecting essential genes. We also perform positive selection CRISPRa screens and demonstrate that Calabrese outperforms the SAM approach at identifying vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-07901-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303322PMC
December 2018

DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells.

Nature 2018 11 31;563(7732):522-526. Epub 2018 Oct 31.

Division of Radiation and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0670-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155769PMC
November 2018

Improved estimation of cancer dependencies from large-scale RNAi screens using model-based normalization and data integration.

Nat Commun 2018 11 2;9(1):4610. Epub 2018 Nov 2.

Broad Institute of MIT and Harvard, Cambridge, 02142, MA, USA.

The availability of multiple datasets comprising genome-scale RNAi viability screens in hundreds of diverse cancer cell lines presents new opportunities for understanding cancer vulnerabilities. Integrated analyses of these data to assess differential dependency across genes and cell lines are challenging due to confounding factors such as batch effects and variable screen quality, as well as difficulty assessing gene dependency on an absolute scale. To address these issues, we incorporated cell line screen-quality parameters and hierarchical Bayesian inference into DEMETER2, an analytical framework for analyzing RNAi screens ( https://depmap.org/R2-D2 ). This model substantially improves estimates of gene dependency across a range of performance measures, including identification of gold-standard essential genes and agreement with CRISPR/Cas9-based viability screens. It also allows us to integrate information across three large RNAi screening datasets, providing a unified resource representing the most extensive compilation of cancer cell line genetic dependencies to date.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-06916-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214982PMC
November 2018
-->