Publications by authors named "Michael S Lawrence"

102 Publications

An extended APOBEC3A mutation signature in cancer.

Nat Commun 2021 03 11;12(1):1602. Epub 2021 Mar 11.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA.

APOBEC mutagenesis, a major driver of cancer evolution, is known for targeting TpC sites in DNA. Recently, we showed that APOBEC3A (A3A) targets DNA hairpin loops. Here, we show that DNA secondary structure is in fact an orthogonal influence on A3A substrate optimality and, surprisingly, can override the TpC sequence preference. VpC (non-TpC) sites in optimal hairpins can outperform TpC sites as mutational hotspots. This expanded understanding of APOBEC mutagenesis illuminates the genomic Twin Paradox, a puzzling pattern of closely spaced mutation hotspots in cancer genomes, in which one is a canonical TpC site but the other is a VpC site, and double mutants are seen only in trans, suggesting a two-hit driver event. Our results clarify this paradox, revealing that both hotspots in these twins are optimal A3A substrates. Our findings reshape the notion of a mutation signature, highlighting the additive roles played by DNA sequence and DNA structure.
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http://dx.doi.org/10.1038/s41467-021-21891-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952602PMC
March 2021

Spectrum of Mechanisms of Resistance to Crizotinib and Lorlatinib in Fusion-Positive Lung Cancer.

Clin Cancer Res 2021 May 8;27(10):2899-2909. Epub 2021 Mar 8.

Department of Medicine, Massachusetts General Hospital Cancer Center, Boston, Massachusetts.

Purpose: Current standard initial therapy for advanced, ROS proto-oncogene 1, receptor tyrosine kinase fusion ()-positive (ROS1) non-small cell lung cancer (NSCLC) is crizotinib or entrectinib. Lorlatinib, a next-generation anaplastic lymphoma kinase/ROS1 inhibitor, recently demonstrated efficacy in ROS1 NSCLC, including in crizotinib-pretreated patients. However, mechanisms of lorlatinib resistance in ROS1 disease remain poorly understood. Here, we assessed mechanisms of resistance to crizotinib and lorlatinib.

Experimental Design: Biopsies from patients with ROS1 NSCLC progressing on crizotinib or lorlatinib were profiled by genetic sequencing.

Results: From 55 patients, 47 post-crizotinib and 32 post-lorlatinib biopsies were assessed. Among 42 post-crizotinib and 28 post-lorlatinib biopsies analyzed at distinct timepoints, mutations were identified in 38% and 46%, respectively. G2032R was the most commonly occurring mutation in approximately one third of cases. Additional mutations included D2033N (2.4%) and S1986F (2.4%) post-crizotinib and L2086F (3.6%), G2032R/L2086F (3.6%), G2032R/S1986F/L2086F (3.6%), and S1986F/L2000V (3.6%) post-lorlatinib. Structural modeling predicted ROS1 causes steric interference to lorlatinib, crizotinib, and entrectinib, while it may accommodate cabozantinib. In Ba/F3 models, ROS1, ROS1, and ROS1 were refractory to lorlatinib but sensitive to cabozantinib. A patient with disease progression on crizotinib and lorlatinib and L2086F received cabozantinib for nearly 11 months with disease control. Among lorlatinib-resistant biopsies, we also identified amplification (4%), G12C (4%), amplification (4%), mutation (4%), and mutation (4%).

Conclusions: mutations mediate resistance to crizotinib and lorlatinib in more than one third of cases, underscoring the importance of developing next-generation ROS1 inhibitors with potency against these mutations, including G2032R and L2086F. Continued efforts are needed to elucidate ROS1-independent resistance mechanisms.
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http://dx.doi.org/10.1158/1078-0432.CCR-21-0032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127383PMC
May 2021

HPV+ oropharyngeal squamous cell carcinomas from patients with two tumors display synchrony of viral genomes yet discordant mutational profiles and signatures.

Carcinogenesis 2021 02;42(1):14-20

Department of Otolaryngology, University of Pittsburgh, Pittsburgh, PA, USA.

Human papillomavirus (HPV) positive oropharyngeal squamous cell carcinoma (HPV + OPSCC) is increasing in prevalence in the USA, as are cases of patients with multiple HPV + OPSCCs (mHPV + OPSCC). mHPV + OPSCCs present a unique opportunity to examine HPV + OPSCC mutation acquisition and evolution. We performed sequencing of the viral genome, somatic exome and somatic transcriptome from 8 patients each with 2 spatially distinct HPV + OPSCCs, and 37 'traditional' HPV + OPSCCs to first address if paired tumors are caused by the same viral isolate and next, if acquired alterations, and the underlying processes driving mutagenesis, are shared within pairs. All tumor pairs contained viral genomes from the same HPV type 16 sublineage and differed by 0-2 clonal single nucleotide polymorphisms (SNPs), suggesting infection with the same viral isolate. Despite this, there was significant discordance in expression profiles, mutational burden and mutational profiles between tumors in a pair, with only two pairs sharing any overlapping mutations (3/3343 variants). Within tumor pairs there was a striking discrepancy of mutational signatures, exemplified by no paired tumors sharing high APOBEC mutational burden. Here, leveraging mHPV + OPSCCs as a model system to study mutation acquisition in virally mediated tumors, in which the germline, environmental exposures, immune surveillance and tissue/organ type were internally controlled, we demonstrate that despite infection by the same viral isolate, paired mHPV + OPSCCs develop drastically different somatic alterations and even more strikingly, appear to be driven by disparate underlying mutational processes. Thus, despite a common starting point, HPV + OPSCCs evolve through variable mutational processes with resultant stochastic mutational profiles.
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http://dx.doi.org/10.1093/carcin/bgaa111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8014522PMC
February 2021

Subependymal giant cell astrocytomas are characterized by mTORC1 hyperactivation, a very low somatic mutation rate, and a unique gene expression profile.

Mod Pathol 2021 02 13;34(2):264-279. Epub 2020 Oct 13.

Cancer Genetics Laboratory, Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

Subependymal giant-cell astrocytomas (SEGAs) are slow-growing brain tumors that are a hallmark feature seen in 5-10% of patients with Tuberous Sclerosis Complex (TSC). Though histologically benign, they can cause serious neurologic symptoms, leading to death if untreated. SEGAs consistently show biallelic loss of TSC1 or TSC2. Herein, we aimed to define other somatic events beyond TSC1/TSC2 loss and identify potential transcriptional drivers that contribute to SEGA formation. Paired tumor-normal whole-exome sequencing was performed on 21 resected SEGAs from 20 TSC patients. Pathogenic variants in TSC1/TSC2 were identified in 19/21 (90%) SEGAs. Copy neutral loss of heterozygosity (size range: 2.2-46 Mb) was seen in 76% (16/21) of SEGAs (44% chr9q and 56% chr16p). An average of 1.4 other somatic variants (range 0-7) per tumor were identified, unlikely of pathogenic significance. Whole transcriptome RNA-sequencing analyses revealed 190 common differentially expressed genes in SEGA (n = 16, 13 from a prior study) in pairwise comparison to each of: low grade diffuse gliomas (n = 530) and glioblastoma (n = 171) from The Cancer Genome Atlas (TCGA) consortium, ganglioglioma (n = 10), TSC cortical tubers (n = 15), and multiple normal tissues. Among these, homeobox transcription factors (TFs) HMX3, HMX2, VAX1, SIX3; and TFs IRF6 and EOMES were all expressed >12-fold higher in SEGAs (FDR/q-value < 0.05). Immunohistochemistry supported the specificity of IRF6, VAX1, SIX3 for SEGAs in comparison to other tumor entities and normal brain. We conclude that SEGAs have an extremely low somatic mutation rate, suggesting that TSC1/TSC2 loss is sufficient to drive tumor growth. The unique and highly expressed SEGA-specific TFs likely reflect the neuroepithelial cell of origin, and may also contribute to the transcriptional and epigenetic state that enables SEGA growth following two-hit loss of TSC1 or TSC2 and mTORC1 activation.
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http://dx.doi.org/10.1038/s41379-020-00659-9DOI Listing
February 2021

Small cell transformation of fusion-positive lung cancer resistant to ROS1 inhibition.

NPJ Precis Oncol 2020 3;4:21. Epub 2020 Aug 3.

Department of Medicine, Massachusetts General Hospital, Boston, MA USA.

Histologic transformation from non-small cell to small cell lung cancer has been reported as a resistance mechanism to targeted therapy in -mutant and fusion-positive lung cancers. Whether small cell transformation occurs in other oncogene-driven lung cancers remains unknown. Here we analyzed the genomic landscape of two pre-mortem and 11 post-mortem metastatic tumors collected from an advanced, fusion-positive lung cancer patient, who had received sequential ROS1 inhibitors. Evidence of small cell transformation was observed in all metastatic sites at autopsy, with inactivation of and , and loss of fusion expression. Whole-exome sequencing revealed minimal mutational and copy number heterogeneity, suggestive of "hard" clonal sweep. Patient-derived models generated from autopsy retained features consistent with small cell lung cancer and demonstrated resistance to ROS1 inhibitors. This case supports small cell transformation as a recurring resistance mechanism, and underscores the importance of elucidating its biology to expand therapeutic opportunities.
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http://dx.doi.org/10.1038/s41698-020-0127-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7400592PMC
August 2020

Identification of Somatically Acquired Mutations by cfDNA Analysis in Patients with Metastatic Breast Cancer.

Clin Cancer Res 2020 09 22;26(18):4852-4862. Epub 2020 Jun 22.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts.

Purpose: Plasma genotyping may identify mutations in potentially "actionable" cancer genes, such as , but their clinical significance is not well-defined. We evaluated the characteristics of somatically acquired mutations in patients with metastatic breast cancer (MBC).

Experimental Design: Patients with MBC undergoing routine cell-free DNA (cfDNA) next-generation sequencing (73-gene panel) before starting a new therapy were included. Somatic mutations were classified as known germline pathogenic mutations or novel variants, and linked to clinicopathologic characteristics. The effect of the PARP inhibitor, olaparib, was assessed , using cultured circulating tumor cells (CTCs) from a patient with a somatically acquired mutation and a second patient with an acquired mutation.

Results: Among 215 patients with MBC, 29 (13.5%) had somatic cfDNA mutations [nine (4%) known germline pathogenic and rest (9%) novel variants]. Known germline pathogenic mutations were common in younger patients ( = 0.008), those with triple-negative disease ( = 0.022), and they were more likely to be protein-truncating alterations and be associated with mutations. Functional analysis of a CTC culture harboring a somatic mutation demonstrated high sensitivity to PARP inhibition, while another CTC culture harboring a somatic mutation showed no differential sensitivity. Across the entire cohort, APOBEC mutational signatures (COSMIC Signatures 2 and 13) and the "BRCA" mutational signature (COSMIC Signature 3) were present in mutant and wild-type cases, demonstrating the high mutational burden associated with advanced MBC.

Conclusions: Somatic mutations are readily detectable in MBC by cfDNA analysis, and may be present as both known germline pathogenic and novel variants.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-0638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501190PMC
September 2020

Quantification of ongoing APOBEC3A activity in tumor cells by monitoring RNA editing at hotspots.

Nat Commun 2020 06 12;11(1):2971. Epub 2020 Jun 12.

Department of Biological Chemistry, Center for Epigenetics and Metabolism, Chao Family Comprehensive Cancer Center, University of California Irvine, Irvine, CA, USA.

APOBEC3A is a cytidine deaminase driving mutagenesis, DNA replication stress and DNA damage in cancer cells. While the APOBEC3A-induced vulnerability of cancers offers an opportunity for therapy, APOBEC3A protein and mRNA are difficult to quantify in tumors due to their low abundance. Here, we describe a quantitative and sensitive assay to measure the ongoing activity of APOBEC3A in tumors. Using hotspot RNA mutations identified from APOBEC3A-positive tumors and droplet digital PCR, we develop an assay to quantify the RNA-editing activity of APOBEC3A. This assay is superior to APOBEC3A protein- and mRNA-based assays in predicting the activity of APOBEC3A on DNA. Importantly, we demonstrate that the RNA mutation-based APOBEC3A assay is applicable to clinical samples from cancer patients. Our study presents a strategy to follow the dysregulation of APOBEC3A in tumors, providing opportunities to investigate the role of APOBEC3A in tumor evolution and to target the APOBEC3A-induced vulnerability in therapy.
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http://dx.doi.org/10.1038/s41467-020-16802-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293259PMC
June 2020

MET Alterations Are a Recurring and Actionable Resistance Mechanism in ALK-Positive Lung Cancer.

Clin Cancer Res 2020 06 21;26(11):2535-2545. Epub 2020 Feb 21.

Massachusetts General Hospital Cancer Center and Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts.

Purpose: Most -positive lung cancers will develop ALK-independent resistance after treatment with next-generation ALK inhibitors. amplification has been described in patients progressing on ALK inhibitors, but frequency of this event has not been comprehensively assessed.

Experimental Design: We performed FISH and/or next-generation sequencing on 207 posttreatment tissue ( = 101) or plasma ( = 106) specimens from patients with ALK-positive lung cancer to detect genetic alterations. We evaluated ALK inhibitor sensitivity in cell lines with alterations and assessed antitumor activity of ALK/MET blockade in ALK-positive cell lines and 2 patients with MET-driven resistance.

Results: amplification was detected in 15% of tumor biopsies from patients relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients treated with a second-generation ALK inhibitor in the first-line setting were more likely to develop amplification than those who had received next-generation ALK inhibitors after crizotinib ( = 0.019). Two tumor specimens harbored an identical rearrangement, one of which had concurrent amplification. Expressing in the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was reversed with dual ALK/MET inhibition. MET inhibition resensitized a patient-derived cell line harboring both and amplification to ALK inhibitors. Two patients with ALK-positive lung cancer and acquired alterations achieved rapid responses to ALK/MET combination therapy.

Conclusions: Treatment with next-generation ALK inhibitors, particularly in the first-line setting, may lead to MET-driven resistance. Patients with acquired alterations may derive clinical benefit from therapies that target both ALK and MET.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269872PMC
June 2020

A post-transcriptional program of chemoresistance by AU-rich elements and TTP in quiescent leukemic cells.

Genome Biol 2020 02 10;21(1):33. Epub 2020 Feb 10.

Massachusetts General Hospital Cancer Center, Harvard Medical School, 185 Cambridge St, CPZN4202, Boston, MA, 02114, USA.

Background: Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown.

Results: We induce chemoresistant and G0 leukemic cells by serum starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the upregulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, Tristetraprolin (TTP) in G0. This permits expression of ARE mRNAs that promote chemoresistance. Conversely, inhibition of TTP phosphorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE-bearing TNFα and DUSP1 mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα prior to or along with chemotherapy substantially reduces chemoresistance in primary leukemic cells ex vivo and in vivo.

Conclusions: These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE-bearing mRNAs that promote chemoresistance. By disrupting this pathway, we develop an effective combination therapy against chemosurvival.
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http://dx.doi.org/10.1186/s13059-020-1936-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7011231PMC
February 2020

Analyses of non-coding somatic drivers in 2,658 cancer whole genomes.

Nature 2020 02 5;578(7793):102-111. Epub 2020 Feb 5.

Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA.

The discovery of drivers of cancer has traditionally focused on protein-coding genes. Here we present analyses of driver point mutations and structural variants in non-coding regions across 2,658 genomes from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). For point mutations, we developed a statistically rigorous strategy for combining significance levels from multiple methods of driver discovery that overcomes the limitations of individual methods. For structural variants, we present two methods of driver discovery, and identify regions that are significantly affected by recurrent breakpoints and recurrent somatic juxtapositions. Our analyses confirm previously reported drivers, raise doubts about others and identify novel candidates, including point mutations in the 5' region of TP53, in the 3' untranslated regions of NFKBIZ and TOB1, focal deletions in BRD4 and rearrangements in the loci of AKR1C genes. We show that although point mutations and structural variants that drive cancer are less frequent in non-coding genes and regulatory sequences than in protein-coding genes, additional examples of these drivers will be found as more cancer genomes become available.
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http://dx.doi.org/10.1038/s41586-020-1965-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054214PMC
February 2020

Pathway and network analysis of more than 2500 whole cancer genomes.

Nat Commun 2020 02 5;11(1):729. Epub 2020 Feb 5.

Department of Computer Science, Princeton University, Princeton, NJ, 08540, USA.

The catalog of cancer driver mutations in protein-coding genes has greatly expanded in the past decade. However, non-coding cancer driver mutations are less well-characterized and only a handful of recurrent non-coding mutations, most notably TERT promoter mutations, have been reported. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancer across 38 tumor types, we perform multi-faceted pathway and network analyses of non-coding mutations across 2583 whole cancer genomes from 27 tumor types compiled by the ICGC/TCGA PCAWG project that was motivated by the success of pathway and network analyses in prioritizing rare mutations in protein-coding genes. While few non-coding genomic elements are recurrently mutated in this cohort, we identify 93 genes harboring non-coding mutations that cluster into several modules of interacting proteins. Among these are promoter mutations associated with reduced mRNA expression in TP53, TLE4, and TCF4. We find that biological processes had variable proportions of coding and non-coding mutations, with chromatin remodeling and proliferation pathways altered primarily by coding mutations, while developmental pathways, including Wnt and Notch, altered by both coding and non-coding mutations. RNA splicing is primarily altered by non-coding mutations in this cohort, and samples containing non-coding mutations in well-known RNA splicing factors exhibit similar gene expression signatures as samples with coding mutations in these genes. These analyses contribute a new repertoire of possible cancer genes and mechanisms that are altered by non-coding mutations and offer insights into additional cancer vulnerabilities that can be investigated for potential therapeutic treatments.
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http://dx.doi.org/10.1038/s41467-020-14367-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002574PMC
February 2020

Epithelial to mesenchymal plasticity and differential response to therapies in pancreatic ductal adenocarcinoma.

Proc Natl Acad Sci U S A 2019 12 16. Epub 2019 Dec 16.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02114;

Transcriptional profiling has defined pancreatic ductal adenocarcinoma (PDAC) into distinct subtypes with the majority being classical epithelial (E) or quasi-mesenchymal (QM). Despite clear differences in clinical behavior, growing evidence indicates these subtypes exist on a continuum with features of both subtypes present and suggestive of interconverting cell states. Here, we investigated the impact of different therapies being evaluated in PDAC on the phenotypic spectrum of the E/QM state. We demonstrate using RNA-sequencing and RNA-in situ hybridization (RNA-ISH) that FOLFIRINOX combination chemotherapy induces a common shift of both E and QM PDAC toward a more QM state in cell lines and patient tumors. In contrast, Vitamin D, another drug under clinical investigation in PDAC, induces distinct transcriptional responses in each PDAC subtype, with augmentation of the baseline E and QM state. Importantly, this translates to functional changes that increase metastatic propensity in QM PDAC, but decrease dissemination in E PDAC in vivo models. These data exemplify the importance of both the initial E/QM subtype and the plasticity of E/QM states in PDAC in influencing response to therapy, which highlights their relevance in guiding clinical trials.
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http://dx.doi.org/10.1073/pnas.1914915116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936349PMC
December 2019

Histone Lysine Methylation Dynamics Control DNA Copy-Number Amplification.

Cancer Discov 2020 02 27;10(2):306-325. Epub 2019 Nov 27.

Department of Medicine, Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, Massachusetts.

Acquired chromosomal DNA copy gains are a feature of many tumors; however, the mechanisms that underpin oncogene amplification are poorly understood. Recent studies have begun to uncover the importance of epigenetic states and histone lysine methyltransferases (KMT) and demethylases (KDM) in regulating transient site-specific DNA copy-number gains (TSSG). In this study, we reveal a critical interplay between a myriad of lysine methyltransferases and demethylases in modulating H3K4/9/27 methylation balance to control extrachromosomal amplification of the oncogene. This study further establishes that cellular signals (hypoxia and EGF) are able to directly promote amplification through modulation of the enzymes controlling copy gains. Moreover, we demonstrate that chemical inhibitors targeting specific KMTs and KDMs are able to promote or block extrachromosomal amplification, which identifies potential therapeutic strategies for controlling copy-number heterogeneity in cancer, and, in turn, drug response. SIGNIFICANCE: This study identifies a network of epigenetic factors and cellular signals that directly control DNA amplification. We demonstrate that chemical inhibitors targeting enzymes controlling this amplification can be used to rheostat copy number, which uncovers therapeutic opportunities for controlling DNA amplification heterogeneity and the associated drug response..
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http://dx.doi.org/10.1158/2159-8290.CD-19-0463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271978PMC
February 2020

Passenger Hotspot Mutations in Cancer.

Cancer Cell 2019 09;36(3):288-301.e14

The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Center for Cancer Research, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115, USA; Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA. Electronic address:

Current statistical models for assessing hotspot significance do not properly account for variation in site-specific mutability, thereby yielding many false-positives. We thus (i) detail a Log-normal-Poisson (LNP) background model that accounts for this variability in a manner consistent with models of mutagenesis; (ii) use it to show that passenger hotspots arise from all common mutational processes; and (iii) apply it to a ∼10,000-patient cohort to nominate driver hotspots with far fewer false-positives compared with conventional methods. Overall, we show that many cancer hotspot mutations recurring at the same genomic site across multiple tumors are actually passenger events, recurring at inherently mutable genomic sites under no positive selection.
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http://dx.doi.org/10.1016/j.ccell.2019.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371346PMC
September 2019

GTP-Dependent Formation of Multimeric G-Quadruplexes.

ACS Chem Biol 2019 09 5;14(9):1951-1963. Epub 2019 Sep 5.

Institute of Organic Chemistry and Biochemistry ASCR , Prague 166 10 , Czech Republic.

G-Quadruplexes are noncanonical nucleic acid structures made up of stacked guanosine tetrads connected by short loops. They are frequently used building blocks in synthetic biology and thought to play widespread biological roles. Multimerization can change the functional properties of G-quadruplexes, and understanding the factors that modulate this process remains an important goal. Here, we report the discovery of a novel mechanism by which the formation of multimeric G-quadruplexes can be controlled using GTP. We show that GTP likely inhibits multimer formation by becoming incorporated into a tetrad in the monomeric form of the structure and define the sequence requirements of G-quadruplexes that form GTP-dependent structures. These experiments provide new insights into the small molecule control of G-quadruplex multimerization. They also suggest possible roles for GTP-dependent multimeric G-quadruplexes in both synthetic and natural biological systems.
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http://dx.doi.org/10.1021/acschembio.9b00428DOI Listing
September 2019

Genome-wide mapping of regions preferentially targeted by the human DNA-cytosine deaminase APOBEC3A using uracil-DNA pulldown and sequencing.

J Biol Chem 2019 10 19;294(41):15037-15051. Epub 2019 Aug 19.

Department of Chemistry, Wayne State University, Detroit, Michigan 48202

Activation-induced deaminase (AID) and apolipoprotein B mRNA-editing enzyme catalytic subunit (APOBEC) enzymes convert cytosines to uracils, creating signature mutations that have been used to predict sites targeted by these enzymes. Mutation-based targeting maps are distorted by the error-prone or error-free repair of these uracils and by selection pressures. To directly map uracils created by AID/APOBEC enzymes, here we used uracil-DNA glycosylase and an alkoxyamine to covalently tag and sequence uracil-containing DNA fragments (UPD-Seq). We applied this technique to the genome of repair-defective, APOBEC3A-expressing bacterial cells and created a uracilation genome map, uracilome. The peak uracilated regions were in the 5'-ends of genes and operons mainly containing tRNA genes and a few protein-coding genes. We validated these findings through deep sequencing of pulldown regions and whole-genome sequencing of independent clones. The peaks were not correlated with high transcription rates or stable RNA:DNA hybrid formation. We defined the uracilation index (UI) as the frequency of occurrence of TT in UPD-Seq reads at different original TC dinucleotides. Genome-wide UI calculation confirmed that APOBEC3A modifies cytosines in the lagging-strand template during replication and in short hairpin loops. APOBEC3A's preference for tRNA genes was observed previously in yeast, and an analysis of human tumor sequences revealed that in tumors with a high percentage of APOBEC3 signature mutations, the frequency of tRNA gene mutations was much higher than in the rest of the genome. These results identify multiple causes underlying selection of cytosines by APOBEC3A for deamination, and demonstrate the utility of UPD-Seq.
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http://dx.doi.org/10.1074/jbc.RA119.008053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791317PMC
October 2019

Combination Olaparib and Temozolomide in Relapsed Small-Cell Lung Cancer.

Cancer Discov 2019 10 15;9(10):1372-1387. Epub 2019 Aug 15.

Massachusetts General Hospital Cancer Center, Boston, Massachusetts.

Small-cell lung cancer (SCLC) is an aggressive malignancy in which inhibitors of PARP have modest single-agent activity. We performed a phase I/II trial of combination olaparib tablets and temozolomide (OT) in patients with previously treated SCLC. We established a recommended phase II dose of olaparib 200 mg orally twice daily with temozolomide 75 mg/m daily, both on days 1 to 7 of a 21-day cycle, and expanded to a total of 50 patients. The confirmed overall response rate was 41.7% (20/48 evaluable); median progression-free survival was 4.2 months [95% confidence interval (CI), 2.8-5.7]; and median overall survival was 8.5 months (95% CI, 5.1-11.3). Patient-derived xenografts (PDX) from trial patients recapitulated clinical OT responses, enabling a 32-PDX coclinical trial. This revealed a correlation between low basal expression of inflammatory-response genes and cross-resistance to both OT and standard first-line chemotherapy (etoposide/platinum). These results demonstrate a promising new therapeutic strategy in SCLC and uncover a molecular signature of those tumors most likely to respond. SIGNIFICANCE: We demonstrate substantial clinical activity of combination olaparib/temozolomide in relapsed SCLC, revealing a promising new therapeutic strategy for this highly recalcitrant malignancy. Through an integrated coclinical trial in PDXs, we then identify a molecular signature predictive of response to OT, and describe the common molecular features of cross-resistant SCLC...
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http://dx.doi.org/10.1158/2159-8290.CD-19-0582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319046PMC
October 2019

Blood-based monitoring identifies acquired and targetable driver mutations in endocrine-resistant metastatic breast cancer.

NPJ Precis Oncol 2019 16;3:18. Epub 2019 Jul 16.

1Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129 USA.

Plasma genotyping identifies potentially actionable mutations at variable mutant allele frequencies, often admixed with multiple subclonal variants, highlighting the need for their clinical and functional validation. We prospectively monitored plasma genotypes in 143 women with endocrine-resistant metastatic breast cancer (MBC), identifying multiple novel mutations including mutations (8.4%), albeit at different frequencies highlighting clinical heterogeneity. To evaluate functional significance, we established ex vivo culture from circulating tumor cells (CTCs) from a patient with -mutant MBC, which revealed resistance to multiple targeted therapies including endocrine and CDK 4/6 inhibitors, but high sensitivity to neratinib (IC50: 0.018 μM). Immunoblotting analysis of the -mutant CTC culture line revealed high levels of HER2 expression at baseline were suppressed by neratinib, which also abrogated downstream signaling, highlighting oncogenic dependency with HER2 mutation. Furthermore, treatment of an index patient with -mutant MBC with the irreversible HER2 inhibitor neratinib resulted in significant clinical response, with complete molecular resolution of two distinct clonal mutations, with persistence of other passenger subclones, confirming HER2 alteration as a driver mutation. Thus, driver mutant alleles that emerge during blood-based monitoring of endocrine-resistant MBC confer novel therapeutic vulnerability, and ex vivo expansion of viable CTCs from the blood circulation may broadly complement plasma-based mutational analysis in MBC.
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http://dx.doi.org/10.1038/s41698-019-0090-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635494PMC
July 2019

Passenger hotspot mutations in cancer driven by APOBEC3A and mesoscale genomic features.

Science 2019 06;364(6447)

Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA.

Cancer drivers require statistical modeling to distinguish them from passenger events, which accumulate during tumorigenesis but provide no fitness advantage to cancer cells. The discovery of driver genes and mutations relies on the assumption that exact positional recurrence is unlikely by chance; thus, the precise sharing of mutations across patients identifies drivers. Examining the mutation landscape in cancer genomes, we found that many recurrent cancer mutations previously designated as drivers are likely passengers. Our integrated bioinformatic and biochemical analyses revealed that these passenger hotspot mutations arise from the preference of APOBEC3A, a cytidine deaminase, for DNA stem-loops. Conversely, recurrent APOBEC-signature mutations not in stem-loops are enriched in well-characterized driver genes and may predict new drivers. This demonstrates that mesoscale genomic features need to be integrated into computational models aimed at identifying mutations linked to diseases.
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http://dx.doi.org/10.1126/science.aaw2872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731024PMC
June 2019

Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer.

Cell 2019 06 30;178(1):160-175.e27. Epub 2019 May 30.

Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.
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http://dx.doi.org/10.1016/j.cell.2019.05.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697165PMC
June 2019

Next-generation characterization of the Cancer Cell Line Encyclopedia.

Nature 2019 05 8;569(7757):503-508. Epub 2019 May 8.

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

Large panels of comprehensively characterized human cancer models, including the Cancer Cell Line Encyclopedia (CCLE), have provided a rigorous framework with which to study genetic variants, candidate targets, and small-molecule and biological therapeutics and to identify new marker-driven cancer dependencies. To improve our understanding of the molecular features that contribute to cancer phenotypes, including drug responses, here we have expanded the characterizations of cancer cell lines to include genetic, RNA splicing, DNA methylation, histone H3 modification, microRNA expression and reverse-phase protein array data for 1,072 cell lines from individuals of various lineages and ethnicities. Integration of these data with functional characterizations such as drug-sensitivity, short hairpin RNA knockdown and CRISPR-Cas9 knockout data reveals potential targets for cancer drugs and associated biomarkers. Together, this dataset and an accompanying public data portal provide a resource for the acceleration of cancer research using model cancer cell lines.
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http://dx.doi.org/10.1038/s41586-019-1186-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697103PMC
May 2019

A Code of Mono-phosphorylation Modulates the Function of RB.

Mol Cell 2019 03 30;73(5):985-1000.e6. Epub 2019 Jan 30.

Massachusetts General Hospital Cancer Center and Harvard Medical School, Building 149 13th Street, Charlestown, MA 02129, USA. Electronic address:

Hyper-phosphorylation of RB controls its interaction with E2F and inhibits its tumor suppressor properties. However, during G1 active RB can be mono-phosphorylated on any one of 14 CDK phosphorylation sites. Here, we used quantitative proteomics to profile protein complexes formed by each mono-phosphorylated RB isoform (mP-RB) and identified the associated transcriptional outputs. The results show that the 14 sites of mono-phosphorylation co-ordinate RB's interactions and confer functional specificity. All 14 mP-RBs interact with E2F/DP proteins, but they provide different shades of E2F regulation. RB mono-phosphorylation at S811, for example, alters RB transcriptional activity by promoting its association with NuRD complexes. The greatest functional differences between mP-RBs are evident beyond the cell cycle machinery. RB mono-phosphorylation at S811 or T826 stimulates the expression of oxidative phosphorylation genes, increasing cellular oxygen consumption. These results indicate that RB activation signals are integrated in a phosphorylation code that determines the diversity of RB activity.
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http://dx.doi.org/10.1016/j.molcel.2019.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6424368PMC
March 2019

Cross-talk between Lysine-Modifying Enzymes Controls Site-Specific DNA Amplifications.

Cell 2018 08 26;174(4):803-817.e16. Epub 2018 Jul 26.

Massachusetts General Hospital Cancer Center and Department of Medicine, Harvard Medical School, 13(th) Street, Charlestown, MA 02129, USA. Electronic address:

Acquired chromosomal DNA amplifications are features of many tumors. Although overexpression and stabilization of the histone H3 lysine 9/36 (H3K9/36) tri-demethylase KDM4A generates transient site-specific copy number gains (TSSGs), additional mechanisms directly controlling site-specific DNA copy gains are not well defined. In this study, we uncover a collection of H3K4-modifying chromatin regulators that function with H3K9 and H3K36 regulators to orchestrate TSSGs. Specifically, the H3K4 tri-demethylase KDM5A and specific COMPASS/KMT2 H3K4 methyltransferases modulate different TSSG loci through H3K4 methylation states and KDM4A recruitment. Furthermore, a distinct chromatin modifier network, MLL1-KDM4B-KDM5B, controls copy number regulation at a specific genomic locus in a KDM4A-independent manner. These pathways comprise an epigenetic addressing system for defining site-specific DNA rereplication and amplifications.
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http://dx.doi.org/10.1016/j.cell.2018.06.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6212369PMC
August 2018

Sequential ALK Inhibitors Can Select for Lorlatinib-Resistant Compound Mutations in ALK-Positive Lung Cancer.

Cancer Discov 2018 06 12;8(6):714-729. Epub 2018 Apr 12.

Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts.

The cornerstone of treatment for advanced ALK-positive lung cancer is sequential therapy with increasingly potent and selective ALK inhibitors. The third-generation ALK inhibitor lorlatinib has demonstrated clinical activity in patients who failed previous ALK inhibitors. To define the spectrum of mutations that confer lorlatinib resistance, we performed accelerated mutagenesis screening of Ba/F3 cells expressing EML4-ALK. Under comparable conditions, -ethyl--nitrosourea (ENU) mutagenesis generated numerous crizotinib-resistant but no lorlatinib-resistant clones harboring single mutations. In similar screens with EML4-ALK containing single resistance mutations, numerous lorlatinib-resistant clones emerged harboring compound mutations. To determine the clinical relevance of these mutations, we analyzed repeat biopsies from lorlatinib-resistant patients. Seven of 20 samples (35%) harbored compound mutations, including two identified in the ENU screen. Whole-exome sequencing in three cases confirmed the stepwise accumulation of mutations during sequential treatment. These results suggest that sequential ALK inhibitors can foster the emergence of compound mutations, identification of which is critical to informing drug design and developing effective therapeutic strategies. Treatment with sequential first-, second-, and third-generation ALK inhibitors can select for compound mutations that confer high-level resistance to ALK-targeted therapies. A more efficacious long-term strategy may be up-front treatment with a third-generation ALK inhibitor to prevent the emergence of on-target resistance. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-1256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984716PMC
June 2018

Comprehensive Characterization of Cancer Driver Genes and Mutations.

Cell 2018 04;173(2):371-385.e18

Computational and Systems Biology, Genome Institute of Singapore, Singapore, 138672.

Identifying molecular cancer drivers is critical for precision oncology. Multiple advanced algorithms to identify drivers now exist, but systematic attempts to combine and optimize them on large datasets are few. We report a PanCancer and PanSoftware analysis spanning 9,423 tumor exomes (comprising all 33 of The Cancer Genome Atlas projects) and using 26 computational tools to catalog driver genes and mutations. We identify 299 driver genes with implications regarding their anatomical sites and cancer/cell types. Sequence- and structure-based analyses identified >3,400 putative missense driver mutations supported by multiple lines of evidence. Experimental validation confirmed 60%-85% of predicted mutations as likely drivers. We found that >300 MSI tumors are associated with high PD-1/PD-L1, and 57% of tumors analyzed harbor putative clinically actionable events. Our study represents the most comprehensive discovery of cancer genes and mutations to date and will serve as a blueprint for future biological and clinical endeavors.
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http://dx.doi.org/10.1016/j.cell.2018.02.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6029450PMC
April 2018

Cell-of-Origin Patterns Dominate the Molecular Classification of 10,000 Tumors from 33 Types of Cancer.

Cell 2018 04;173(2):291-304.e6

Van Andel Research Institute, Grand Rapids, MI 49503, USA. Electronic address:

We conducted comprehensive integrative molecular analyses of the complete set of tumors in The Cancer Genome Atlas (TCGA), consisting of approximately 10,000 specimens and representing 33 types of cancer. We performed molecular clustering using data on chromosome-arm-level aneuploidy, DNA hypermethylation, mRNA, and miRNA expression levels and reverse-phase protein arrays, of which all, except for aneuploidy, revealed clustering primarily organized by histology, tissue type, or anatomic origin. The influence of cell type was evident in DNA-methylation-based clustering, even after excluding sites with known preexisting tissue-type-specific methylation. Integrative clustering further emphasized the dominant role of cell-of-origin patterns. Molecular similarities among histologically or anatomically related cancer types provide a basis for focused pan-cancer analyses, such as pan-gastrointestinal, pan-gynecological, pan-kidney, and pan-squamous cancers, and those related by stemness features, which in turn may inform strategies for future therapeutic development.
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http://dx.doi.org/10.1016/j.cell.2018.03.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5957518PMC
April 2018