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.
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.
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.
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.
.
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.
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.
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.
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.
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.
Biopsies from patients with ROS1
NSCLC progressing on crizotinib or lorlatinib were profiled by genetic sequencing.
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%).
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.
Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated ...response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3′ UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.
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•NR4A1 specifically localizes to IEG gene bodies suppressing transcriptional elongation•Acute replication stress triggers NR4A1 release, triggering burst of IEG expression•NR4A1 deletion deregulates expression of the IEG FOS, triggering mitotic catastrophe•The NR4A1-IEG pathway denotes differentiated cancers with a favorable prognosis
Studying tumorigenesis by patient-derived breast cancer cells, Guo et al. identify NR4A1 as a master regulator of replication stress-induced immediate early gene expression through a transcriptional processing checkpoint. Suppression of NR4A1 in cancer cells dependent on this pathway triggers mitotic catastrophe through unregulated expression of the IEG family member FOS.
Analysis of The Cancer Genome Atlas and other published data of head and neck squamous cell carcinoma (HNSCC) reveals somatic alterations of the Hippo-YAP pathway in approximately 50% of HNSCC. ...Better strategies to target the YAP1 transcriptional complex are sought. Here, we show that FAT1, an upstream inhibitor of YAP1, is mutated either by missense or by truncating mutation in 29% of HNSCC. Comprehensive proteomic and drug-screening studies across pan-cancer models confirm that FAT1-mutant HNSCC exhibits selective and higher sensitivity to BRD4 inhibition by JQ1. Epigenomic analysis reveals an active chromatin state in FAT1-mutant HNSCC cells that is driven by the YAP/TAZ transcriptional complex through recruitment of BRD4 to deposit active histone marks, thereby maintaining an oncogenic transcriptional state. This study reveals a detailed cooperative mechanism between YAP1 and BRD4 in HNSCC and suggests a specific therapeutic opportunity for the treatment of this subset of head and neck cancer patients.
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•FAT1 mutation contributes to YAP1 transcriptional activation•FAT1-mutant HNSCC exhibits selective and higher sensitivity to BRD4 inhibition•YAP1/TAZ/TEAD transcriptional complex recruits BRD4 to promote active chromatin state•YAP1 and BRD4 regulate multiple oncogenic transcriptional programs in HNSCC
Chen et al. report that FAT1 mutation contributes to YAP1 transcriptional activation and exhibits selective sensitivity to BRD4 inhibitors. Mechanistically, YAP1-driven oncogenic chromatin state in FAT1-mutant HNSCC is through recruitment of BRD4. This study suggests BRD4 inhibitors as a therapeutic opportunity for FAT1-mutant HNSCC patients.
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.
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.
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.
APOBEC is a mutagenic source in human papillomavirus (HPV)-mediated malignancies, including HPV+ oropharyngeal squamous cell carcinoma (HPV + OPSCC), and in HPV genomes. It is unknown why APOBEC ...mutations predominate in HPV + OPSCC, or if the APOBEC-induced mutations observed in both human cancers and HPV genomes are directly linked. We performed sequencing of host somatic exomes, transcriptomes, and HPV16 genomes from 79 HPV + OPSCC samples, quantifying APOBEC mutational burden and activity in both host and virus. APOBEC was the dominant mutational signature in somatic exomes. In viral genomes, there was a mean of five (range 0-29) mutations per genome. The mean of APOBEC mutations in viral genomes was one (range 0-5). Viral APOBEC mutations, compared to non-APOBEC mutations, were more likely to be low-variant allele fraction mutations, suggesting that APOBEC mutagenesis actively occurrs in viral genomes during infection. HPV16 APOBEC-induced mutation patterns in OPSCC were similar to those previously observed in cervical samples. Paired host and viral analyses revealed that APOBEC-enriched tumor samples had higher viral APOBEC mutation rates (
= 0.028), and APOBEC-associated RNA editing (
= 0.008), supporting the concept that APOBEC mutagenesis in host and viral genomes is directly linked and occurrs during infection. Using paired sequencing of host somatic exomes, transcriptomes, and viral genomes, we demonstrated for the first-time definitive evidence of concordance between tumor and viral APOBEC mutagenesis. This finding provides a missing link connecting APOBEC mutagenesis in host and virus and supports a common mechanism driving APOBEC dysregulation.
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