Molecular Predictors of 3D Morphogenesis by Breast Cancer Cell Lines in 3D Culture

Correlative analysis of molecular markers with phenotypic signatures is the simplest model for hypothesis generation. In this paper, a panel of 24 breast cell lines was grown in 3D culture, their morphology was imaged through phase contrast microscopy, and computational methods were developed to segment and represent each colony at multiple dimensions. Subsequently, subpopulations from these morphological responses were identified through consensus clustering to reveal three clusters of round, grape-like, and stellate phenotypes. In some cases, cell lines with particular pathobiological phenotypes clustered together (e.g., ERBB2 amplified cell lines sharing the same morphometric properties as the grape-like phenotype). Next, associations with molecular features were realized through (i) differential analysis within each morphological cluster, and (ii) regression analysis across the entire panel of cell lines. In both cases, the dominant genes that are predictive of the morphological signatures were identified. Specifically, PPARγ has been associated with the invasive stellate morphological phenotype, which corresponds to triple-negative pathobiology. PPARγ has been validated through two supporting biological assays

Lactate-Mediated Epigenetic Reprogramming Regulates Formation of Human Pancreatic Cancer-Associated Fibroblasts

Even though pancreatic ductal adenocarcinoma (PDAC) is associated with fibrotic stroma, the molecular pathways regulating the formation of cancer associated fibroblasts (CAFs) are not well elucidated. An epigenomic analysis of patient-derived and de-novo generated CAFs demonstrated widespread loss of cytosine methylation that was associated with overexpression of various inflammatory transcripts including CXCR4. Co-culture of neoplastic cells with CAFs led to increased invasiveness that was abrogated by inhibition of CXCR4. Metabolite tracing revealed that lactate produced by neoplastic cells leads to increased production of alpha-ketoglutarate (aKG) within mesenchymal stem cells (MSCs). In turn, aKG mediated activation of the demethylase TET enzyme led to decreased cytosine methylation and increased hydroxymethylation during de novo differentiation of MSCs to CAF. Co-injection of neoplastic cells with TET-deficient MSCs inhibited tumor growth in vivo. Thus, in PDAC, a tumor-mediated lactate flux is associated with widespread epigenomic reprogramming that is seen during CAF formation

Inhibitors of BRAF dimers using an allosteric site

FDA-approved RAF inhibitors poorly inhibit BRAF dimers, which limits their clinical efficacy in tumors expressing BRAFV600E mutant monomers. Here the authors identify FDA-approved Ponatinib as an effective inhibitor of BRAF monomers and dimers and designed PHI1, an inhibitor with a unique mode of action and selectivity for oncogenic BRAF dimers

Initial Whole Genome Sequencing of Plasma Cell Neoplasms in First Responders and Recovery Workers Exposed to the World Trade Center Attack of September 11, 2001

PURPOSE : The World Trade Center (WTC) attack of September 11, 2001 created an unprecedented environmental exposure to known and suspected carcinogens. A higher incidence of multiple myeloma (MM) and precursor disease has been reported among first responders to the WTC disaster compared to the unexposed population (Landgren, 2018). To expand on prior screening studies, and to characterize the genomic impact of the exposure to known and potential carcinogens in the WTC debris, we were motivated to perform whole genome sequencing (WGS) of WTC first responders and recovery workers who were diagnosed with a plasma cell disorder after the attack. PATIENTS AND METHODS: We performed WGS of 9 CD138-positive bone marrow mononuclear samples from patients who were diagnosed with plasma cell disorders after exposure to the WTC disaster: 4 monoclonal gammopathy of undetermined significance (MGUS), 2 smoldering multiple myeloma (SMM), 2 MMs, and 1 patient with plasma cell leukemia (PCL). Eight patients (88%) were first responders and one was a recovery worker. Peripheral blood mononuclear cells were used as normal match. Median coverage for tumor and normal samples was 50.9X (range 47-76) and 37X (range 35-41), respectively. The landscape of genomic drivers and complex structural events was compared to 752 MM patients enrolled in the CoMMpass trial with available whole exome and low-coverage long-insert WGS data (IA15; NCT01454297). To characterize the mutational signature landscape we combined the WTC cohort with 110 whole genomes from 56 patients with multiple myeloma and myeloma precursor disease (Rustad et al. 2020; Landau et al. 2020) and we ran our three-step workflow: de novo extraction (i.e. sigprofiler), assignment, and fitting (i.e. mmsig). To exclude contribution of any environmental agents in the WTC debris with known mutational signatures (Kucab et al., 2019), we ran our fitting algorithm mmsig in each post-WTC case, including and forcing the extraction of these mutational signatures. RESULTS: No significant differences were observed in comparing the post-WTC driver and mutational signatures landscape with 110 previously published WGS from 56 patients with MM and the CoMMpass WGS cohort (n=752). Likewise, we did not observe any new or distinct mutational signatures among WTC-exposed patients. Following forced extraction of 5 mutational signatures associated with environmental agents detected in the WTC debris (e.g. PAHs), we did not find significant contributions from any of these described environmental mutational signatures. To reconstruct the temporal activity of each mutational process we divided all single nucleotide variants into subclonal and clonal. Clonal mutations were further subdivided into duplicated (acquired before a chromosomal gain) and unduplicated (Rustad et al. 2020). WTC-exposed patients had differing patterns in mutational signature timelines of AID and APOBEC activity. Overall, the mutational signature activity over time in post-WTC plasma cell dyscrasia reflects what has been previously observed in multiple myeloma without WTC-exposure (Rustad et al., 2020). Finally, leveraging constant activity of the clock-like single base substitution mutational signatures 1 and 5 over time and our molecular time workflow (Rustad et al., 2020), we estimated the age at which each evaluable patient acquired a tumor-initiating chromosomal gain and found that they were windowed to both pre- and post-WTC exposure across neoplasms (Figure 1). In some cases, clonal multi-chromosomal gain events were acquired decades before both the diagnosis and the WTC exposure. Specifically, of 6 patients with large clonal chromosomal gains, 1 MM case, 1 SMM, and 1 MGUS showed evidence of a pre-existing clone prior to WTC exposure, two MGUS showed evidence of multi-gain events following the exposure, and one MM case had a 1q gain in the same time window as the attack. CONCLUSIONS: Post-WTC plasma cell neoplasms had similar genomic landscapes to non-exposed cases. Although limitations in sample size preclude any definitive conclusions, our findings suggest that the observed increased incidence of plasma cell neoplasms in this population is due to complex and heterogeneous effects of the WTC exposure that may have initiated or contributed to progression of malignancy. The existence of pre-malignant clonal entities at time of WTC exposure may therefore be relevant for future WTC-related study. Figure 1 Disclosures Hultcrantz: Intellisphere LLC: Consultancy; Amgen: Research Funding; Daiichi Sankyo: Research Funding; GSK: Research Funding. Shah:Physicians Education Resource: Honoraria; Celgene/BMS: Research Funding. Iacobuzio-Donahue:BMS: Research Funding. Papaemmanuil:Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Illumina: Consultancy, Honoraria; Kyowa Hakko Kirin: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Prime Oncology: Consultancy, Honoraria; MSKCC: Patents & Royalties. Verma:BMS: Consultancy, Research Funding; acceleron: Consultancy, Honoraria; stelexis: Current equity holder in private company; Janssen: Research Funding; Medpacto: Research Funding. Dogan:National Cancer Institute: Research Funding; EUSA Pharma: Consultancy; Takeda: Consultancy; Seattle Genetics: Consultancy; Corvus Pharmaceuticals: Consultancy; Physicians Education Resource: Consultancy; Roche: Consultancy, Research Funding; AbbVie: Consultancy. Landgren:Celgene: Consultancy, Honoraria, Research Funding; Seattle Genetics: Research Funding; Cellectis: Consultancy, Honoraria; Juno: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Merck: Other; Karyopharma: Research Funding; Janssen: Consultancy, Honoraria, Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Pfizer: Consultancy, Honoraria; Merck: Other; Karyopharma: Research Funding; Binding Site: Consultancy, Honoraria; Takeda: Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Seattle Genetics: Research Funding; Takeda: Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Janssen: Consultancy, Honoraria, Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; Juno: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Adaptive: Consultancy, Honoraria; Cellectis: Consultancy, Honoraria; Glenmark: Consultancy, Honoraria, Research Funding; Binding Site: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Glenmark: Consultancy, Honoraria, Research Funding

Initial Whole-Genome Sequencing of Plasma Cell Neoplasms in First Responders and Recovery Workers Exposed to the World Trade Center Attack of September 11, 2001

The World Trade Center (WTC) attack of September 11, 2001 created an unprecedented environmental exposure to known and suspected carcinogens. High incidence of multiple myeloma and precursor conditions has been reported among first responders to the WTC disaster. To expand on our prior screening studies, and to characterize the genomic impact of the exposure to known and potential carcinogens in the WTC debris, we were motivated to perform whole-genome sequencing (WGS) of WTC first responders and recovery workers who developed a plasma cell disorder after the attack. We performed WGS of nine CD138-positive bone marrow mononuclear samples from patients who were diagnosed with plasma cell disorders after the WTC disaster. No significant differences were observed in comparing the post-WTC driver and mutational signature landscapes with 110 previously published WGSs from 56 patients with multiple myeloma and the CoMMpass WGS cohort ( = 752). Leveraging constant activity of the single-base substitution mutational signatures 1 and 5 over time, we estimated that tumor-initiating chromosomal gains were windowed to both pre- and post-WTC exposure. Although limitations in sample size preclude any definitive conclusions, our findings suggest that the observed increased incidence of plasma cell neoplasms in this population is due to complex and heterogeneous effects of the WTC exposure that may have initiated or contributed to progression of malignancy

High Burden of Clonal Hematopoiesis in First Responders Exposed to the World Trade Center Disaster

Introduction The World Trade Center (WTC) disaster exposed first responders to high levels of aerosolized carcinogens (Lioy et. al. Env. Health Perspect 2002). Clonal hematopoiesis is associated with exposure to smoking and genotoxic stimuli (Jaiswal et. al. NEJM 2014; Genovese et. al. NEJM 2015). We sought to determine its incidence in WTC-exposed first responders. We also assessed the effect of WTC particulate matter (WTC-PM) on genome integrity in vitro, and in murine studies. Methods Deep targeted sequencing was performed on blood collected from 481 first responders (429 WTC-exposed firefighters, 52 WTC-exposed emergency medical service workers) and 52 non-exposed first responders. Samples were analyzed for 237 genes mutated in hematologic malignancies and interpreted using reference databases. Non synonymous somatic mutations were annotated and analyzed. Results In the WTC-exposed cohort, 57 individuals with 66 somatic mutations of expected pathogenic potential were identified (overall prevalence 11.9%). In the non-exposed cohort, only one pathogenic mutation was found in the IDH2 gene (overall prevalence 1.9%). There was a strong association between increasing age and prevalence of mutations in the WTC-exposed cohort (Fig 1A). DNMT3A (16/66), TET2 (7/66), SF3B1 and SRSF2 (3/66 each) were the most common genes identified in the WTC-exposed cohort (Fig 1B). Median VAF was 12% and missense mutations were most frequent alteration. Aging, smoking, DNA repair and alkylating agent exposure related mutational signatures were observed with a cytosine to thymine (C→T) transition being most common. Next, we assessed the effect of WTC-PM on genome integrity and replication in vitro. WTC-PM that was collected in the first three days after 9/11 was used in concentrations mimicking exposure levels. Lymphocytes exposed to WTC-PM demonstrated a significant increase in phosphorylated H2AX foci accumulation, suggesting a DNA damage response (Fig 2). Since common fragile sites (CFSs) detect basal levels of stress in the cell, and activate DNA damage response (DDR), we profiled DNA replication dynamics at CFS-FRA16D at very high resolution using the single molecule analysis of replicated DNA (SMARD) assay. Treatment with WTC-PM significantly altered replication at two common fragile sites (regions 1 and 2 of FRA16D, Fig 3A) with replication pausing being observed at multiple sites (Fig 3B-I, white rectangles). Striking increase in replication initiation was seen, characterized as dormant origins activated to rescue replication pausing (Fig 3E, J). These alterations were accompanied by a corresponding increase in replication speed, conditions that lead to DNA replication errors and mutagenesis (Fig 3F, K). Next, we treated mice with WTC-PM via the oropharyngeal route to mimic first responder exposures, and then harvested and analyzed their bone marrow compartments. Significant expansion of hematopoietic stem cells (Kit+, Sca1+, Lineage-ve, KSL) was seen in WTC-PM treated mice (Fig 4A,B). Whole genome sequencing of sorted stem cells showed a significant increase in non-synonymous SNPs, deletions and indels in the WTC-PM treated samples when compared to control (Fig 4C-E). These genomic alterations were found to occur at low VAF throughout the whole genome, demonstrating widespread genotoxic effects of WTC-PM on hematopoietic stem cells in vivo (Fig 4F). Discussion We report a high burden of mutations in 11.9% (57/481) WTC-exposed first responders compared to the non-exposed cohort (1.9%, 1/52). The frequency of the somatic mutations was many fold higher than in previous studies (Jaiswal et. al. NEJM 2014; Genovese et. al. NEJM, 2015). In the 50-59 year age group, 10% of WTC-exposed individuals carried somatic mutations, compared to the frequency of 2.5% reported by Jaiswal et. al. for the same age group. Despite deeper sequencing performed in our study, the median VAF in our study was 12%, indicating that the difference in technique did not bias our study towards increased detection of small, subclinical clones when compared to previous studies. Furthermore, we demonstrate that WTC-PM can perturb DNA replication and increased genomic instability in vivo, potentially leading to higher burden of clonal hematopoiesis in WTC-exposed first responders. These results demonstrate adverse environmental exposures can be associated with a high rate of clonal hematopoiesis. Disclosures Landgren: Sanofi: Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Theradex: Other: IDMC; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Fletcher:Genoptix/Neogenomics: Employment. Ebert:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.; Celgene: Research Funding; Deerfield: Research Funding. Steidl:GlaxoSmithKline: Research Funding; Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Will:Novartis Pharmaceuticals: Research Funding. Verma:Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding