Invasive mucinous adenocarcinoma (IMA) is a unique subtype of lung adenocarcinoma, characterized genomically by frequent
mutations or specific gene fusions, most commonly involving
. Comprehensive ...analysis of a large series of IMAs using broad DNA- and RNA-sequencing methods is still lacking, and it remains unclear whether molecular subtypes of IMA differ clinicopathologically.
A total of 200 IMAs were analyzed by 410-gene DNA next-generation sequencing (MSK-IMPACT;
= 136) or hotspot 8-oncogene genotyping (
= 64). Driver-negative cases were further analyzed by 62-gene RNA sequencing (MSK-Fusion) and those lacking fusions were further tested by whole-exome sequencing and whole-transcriptome sequencing (WTS).
Combined MSK-IMPACT and MSK-Fusion testing identified mutually exclusive driver alterations in 96% of IMAs, including
mutations (76%),
fusions (7%),
alterations (6%), and other less common events. In addition, WTS identified a novel
fusion (
-
). Overall, targetable gene fusions were identified in 51% of
wild-type IMAs, leading to durable responses to targeted therapy in some patients. Compared with
-mutant IMAs,
-rearranged tumors exhibited several more aggressive characteristics, including worse recurrence-free survival (
< 0.0001).
This is the largest molecular study of IMAs to date, where we demonstrate the presence of a major oncogenic driver in nearly all cases. This study is the first to document more aggressive characteristics of
-rearranged IMAs,
as the third most common alteration, and a novel
fusion in these tumors. Comprehensive molecular testing of
wild-type IMAs that includes fusion testing is essential, given the high prevalence of alterations with established and investigational targeted therapies in this subset.
Despite rapid developments in single cell sequencing, sample-specific batch effects, detection of cell multiplets, and experimental costs remain outstanding challenges. Here, we introduce Cell ...Hashing, where oligo-tagged antibodies against ubiquitously expressed surface proteins uniquely label cells from distinct samples, which can be subsequently pooled. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its original sample, robustly identify cross-sample multiplets, and "super-load" commercial droplet-based systems for significant cost reduction. We validate our approach using a complementary genetic approach and demonstrate how hashing can generalize the benefits of single cell multiplexing to diverse samples and experimental designs.
TRK fusions are found in a variety of cancer types, lead to oncogenic addiction, and strongly predict tumor-agnostic efficacy of TRK inhibition
. With the recent approval of the first selective TRK ...inhibitor, larotrectinib, for patients with any TRK-fusion-positive adult or pediatric solid tumor, to identify mechanisms of treatment failure after initial response has become of immediate therapeutic relevance. So far, the only known resistance mechanism is the acquisition of on-target TRK kinase domain mutations, which interfere with drug binding and can potentially be addressable through second-generation TRK inhibitors
. Here, we report off-target resistance in patients treated with TRK inhibitors and in patient-derived models, mediated by genomic alterations that converge to activate the mitogen-activated protein kinase (MAPK) pathway. MAPK pathway-directed targeted therapy, administered alone or in combination with TRK inhibition, re-established disease control. Experimental modeling further suggests that upfront dual inhibition of TRK and MEK may delay time to progression in cancer types prone to the genomic acquisition of MAPK pathway-activating alterations. Collectively, these data suggest that a subset of patients will develop off-target mechanisms of resistance to TRK inhibition with potential implications for clinical management and future clinical trial design.
Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone post-translational modification (PTM) signatures remains a daunting task in the epigenetics ...field. We introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semisynthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries, once they have been treated with purified chromatin effectors or the combined chromatin recognizing and modifying activities of the nuclear proteome, is followed by multiplexed DNA-barcode sequencing. This ultrasensitive workflow allowed us to collect thousands of biochemical data points revealing the binding preferences of various nuclear factors for PTM patterns and how preexisting PTMs, alone or synergistically, affect further PTM deposition via cross-talk mechanisms. We anticipate that the high throughput and sensitivity of the technology will help accelerate the decryption of the diverse molecular controls that operate at the level of chromatin.
High-throughput single-cell RNA sequencing has transformed our understanding of complex cell populations, but it does not provide phenotypic information such as cell-surface protein levels. Here, we ...describe cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), a method in which oligonucleotide-labeled antibodies are used to integrate cellular protein and transcriptome measurements into an efficient, single-cell readout. CITE-seq is compatible with existing single-cell sequencing approaches and scales readily with throughput increases.
Lineage plasticity, the ability to transdifferentiate among distinct phenotypic identities, facilitates therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon includes ...small cell and squamous cell (LUSC) histologic transformation in the context of acquired resistance to targeted inhibition of driver mutations. LUAD-to-LUSC transdifferentiation, occurring in up to 9% of EGFR-mutant patients relapsed on osimertinib, is associated with notably poor prognosis. We hypothesized that multi-parameter profiling of the components of mixed histology (LUAD/LUSC) tumors could provide insight into factors licensing lineage plasticity between these histologies.
We performed genomic, epigenomics, transcriptomics and protein analyses of microdissected LUAD and LUSC components from mixed histology tumors, pre-/post-transformation tumors and reference non-transformed LUAD and LUSC samples. We validated our findings through genetic manipulation of preclinical models in vitro and in vivo and performed patient-derived xenograft (PDX) treatments to validate potential therapeutic targets in a LUAD PDX model acquiring LUSC features after osimertinib treatment.
Our data suggest that LUSC transdifferentiation is primarily driven by transcriptional reprogramming rather than mutational events. We observed consistent relative upregulation of PI3K/AKT, MYC and PRC2 pathway genes. Concurrent activation of PI3K/AKT and MYC induced squamous features in EGFR-mutant LUAD preclinical models. Pharmacologic inhibition of EZH1/2 in combination with osimertinib prevented relapse with squamous-features in an EGFR-mutant patient-derived xenograft model, and inhibition of EZH1/2 or PI3K/AKT signaling re-sensitized resistant squamous-like tumors to osimertinib.
Our findings provide the first comprehensive molecular characterization of LUSC transdifferentiation, suggesting putative drivers and potential therapeutic targets to constrain or prevent lineage plasticity.
Estrogen receptor alpha (ERα) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic ...targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs. We now identify the induction of an ERα isoform, ERα-LBD, that is encoded by an alternative ESR1 transcript and lacks the activation function and DNA binding domains. Despite lacking the transcriptional activity, ERα-LBD is found to promote breast cancer growth and resistance to the ERα antagonist fulvestrant. ERα-LBD is predominantly localized to the cytoplasm and mitochondria of BC cells and leads to enhanced glycolysis, respiration and stem-like features. Intriguingly, ERα-LBD expression and function does not appear to be restricted to cancers that express full length ERα but also promotes growth of triple-negative breast cancers and ERα-LBD transcript (ESR1-LBD) is also present in BC samples from both ERα(+) and ERα(-) human tumors. These findings point to ERα-LBD as a potential mediator of breast cancer progression and therapy resistance.
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