The identification of novel, oncogenic gene rearrangements in inflammatory myofibroblastic tumor (IMT) demonstrates the potential of next generation sequencing (NGS) platforms for the detection of therapeutically relevant oncogenes across multiple tumor types, but raises significant questions relating to the investigation of targeted therapies in this new era of widespread NGS testing.
With multiple clinical trials underway targeting autophagy against cancer, Yang et al. (1) and Karsli-Uzunbas et al. (2) address important concerns regarding autophagy inhibition in cancer patients using genetically engineered mouse models (GEMMs) that more accurately represent the tumor biology found in human pancreatic and lung cancer patients.
NUT midline carcinoma (NMC) is an aggressive subtype of squamous cell carcinoma that typically harbors BRD4/3-NUT fusion oncoproteins that block differentiation and maintain tumor growth. In 20% of cases NUT is fused to uncharacterized non-BRD gene(s). We established a new patient-derived NMC cell line (1221) and demonstrated that it harbors a novel NSD3-NUT fusion oncogene. We find that NSD3-NUT is both necessary and sufficient for the blockade of differentiation and maintenance of proliferation in NMC cells. NSD3-NUT binds to BRD4, and BRD bromodomain inhibitors induce differentiation and arrest proliferation of 1221 cells. We find further that NSD3 is required for the blockade of differentiation in BRD4-NUT-expressing NMCs. These findings identify NSD3 as a novel critical oncogenic component and potential therapeutic target in NMC.
Loss of PTEN is a common event in many cancers and leads to hyperactivation of the PI 3-K/Akt signaling pathway. The mechanisms by which Akt isoforms mediate signaling to phenotypes associated with PTEN-inactivation in cancer have not been defined. Here we show that Akt2 is exclusively required for PTEN-deficient prostate tumor spheroid maintenance whereas Akt1 is dispensable. shRNA silencing of Akt2 but not Akt1 promotes regression of prostate cancer xenografts. Mechanistically, we show that Akt2 silencing up-regulates p21 and the pro-apoptotic protein Bax and downregulates the insulin-like growth factor receptor-1. We also show that p21 is an effector of Akt2 in mediating prostate tumor maintenance. Moreover, Akt2 is also exclusively required for the maintenance and survival of other PTEN-deficient solid tumors, including breast cancer and glioblastoma. These findings identify a specific function for Akt2 in mediating survival of PTEN-deficient tumors and provide a rationale for developing therapeutics targeting Akt2.
Glioblastomas with EGFR amplification represent approximately 50% of newly diagnosed cases and recent studies have revealed frequent coexistence of multiple EGFR aberrations within the same tumor with implications for mutation cooperation and treatment resistance. However, bulk tumor sequencing studies cannot resolve the patterns of how the multiple EGFR aberrations coexist with other mutations within single tumor cells. Here we applied a population-based single-cell whole genome sequencing methodology to characterize genomic heterogeneity in EGFR amplified glioblastomas. Our analysis effectively identified clonal events, including a novel translocation of a super enhancer to the TERT promoter, as well as subclonal loss-of-heterozygosity and multiple EGFR mutational variants within tumors. Correlating the EGFR mutations onto the cellular hierarchy revealed that EGFR truncation variants (EGFRvII and EGFR Carboxyl-terminal deletions) identified in the bulk tumor segregate into non-overlapping subclonal populations. In vitro and in vivo functional studies show EGFRvII is oncogenic and sensitive to EGFR inhibitors currently in clinical trials. Thus the association between diverse activating mutations in EGFR and other subclonal mutations within a single tumor supports an intrinsic mechanism for proliferative and clonal diversification with broad implications in resistance to treatment.
single-cell sequencing; single-nucleus sequencing; glioblastoma; EGFR; EGFR variant II
Inflammatory myofibroblastic tumor (IMT) is a neoplasm which typically occurs in children. The genetic landscape of this tumor is incompletely understood and therapeutic options are limited. While 50% of IMTs harbor ALK rearrangements, no therapeutic targets have been identified in ALK negative tumors. We report for the first time that IMTs harbor other actionable targets, including ROS1 and PDGFRβ fusions. We detail the case of an 8 year old boy with treatment-refractory ALK negative IMT. Molecular tumor profiling revealed a ROS1 fusion, and he had a dramatic response to the ROS1 inhibitor, crizotinib. This case prompted assessment of a larger series of IMTs. Next generation sequencing revealed that 85% of cases evaluated harbored kinase fusions, involving ALK, ROS1, or PDGFRβ. Our study represents the most comprehensive genetic analysis of IMTs to date and also provides rationale for routine molecular profiling of these tumors to detect therapeutically actionable kinase fusions.
Inflammatory myofibroblastic tumor; ALK; ROS1; PDGFRβ; tyrosine kinase; tyrosine kinase inhibitor; crizotinib; cancer; sarcoma; targeted therapeutics; next generation sequencing; gene rearrangement; gene fusion
Prostate cancer (CaP) is the most prevalent cancer in males and treatment options are limited for advanced forms of the disease. Loss of the PTEN and p53 tumor suppressor genes is commonly observed in CaP, while their compound loss is often observed in advanced CaP. Here we show, that PARP inhibition triggers a p53-dependent cellular senescence in a PTEN-deficient setting in the prostate. Surprisingly, we also find that PARP-induced cellular senescence is morphed into an apoptotic response upon compound loss of PTEN and p53. We further show that superactivation of the pro-survival signalling PI3K-AKT pathway limits the efficacy of a PARP-single-agent treatment, and that PARP and PI3K inhibitors effectively synergize to suppress tumorigenesis in human CaP cell lines and in a Pten/p53 deficient mouse model of advanced CaP. Our findings therefore identify a combinatorial treatment with PARP and PI3K inhibitors as an effective option for PTEN-deficient CaP.
PTEN; Prostate; PARP; PI3K; Senescence
Pancreatic ductal adenocarcinoma is refractory to available therapies. We have previously shown that these tumors have elevated autophagy and inhibition of autophagy leads to decreased tumor growth. Using an autochthonous model of pancreatic cancer driven by oncogenic Kras and the stochastic LOH of p53, we demonstrate that while genetic ablation of autophagy in the pancreas leads to increased tumor initiation, these premalignant lesions are impaired in their ability to progress to invasive cancer, leading to prolonged survival. Additionally, mouse pancreatic cancer cell lines with differing p53 status are all sensitive to pharmacologic and genetic inhibition of autophagy. Lastly, a mouse pre-clinical trial using cohorts of genetically characterized patient derived xenografts treated with hydroxychloroquine showed responses across the collection of tumors. Together our data support the critical role of autophagy in pancreatic cancer and that inhibition of autophagy may have clinical utility in the treatment of these cancers, independent of p53 status.
autophagy; pancreatic cancer; p53; chloroquine; metabolism; Atg5
Macroautophagy (autophagy hereafter) recycles intracellular components to sustain mitochondrial metabolism that promotes the growth, stress tolerance and malignancy of lung cancers, suggesting that autophagy inhibition may have antitumor activity. To assess the functional significance of autophagy in both normal and tumor tissue, we conditionally deleted the essential autophagy gene, autophagy-related-7, Atg7, throughout adult mice. Here we report that systemic ATG7 ablation caused susceptibility to infection and neurodegeneration that limited survival to 2–3 months. Moreover, upon fasting, autophagy-deficient mice suffered fatal hypoglycemia. Prior autophagy ablation did not alter the efficiency of non-small-cell lung cancer (NSCLC) initiation by activation of oncogenic KrasG12D and deletion of the Trp53 tumor suppressor. Acute autophagy ablation in mice with pre-existing NSCLC, however, blocked tumor growth, promoted tumor cell death, and generated more benign disease (oncocytomas). This anti-tumor activity occurred prior to destruction of normal tissues, suggesting that, acute autophagy inhibition may be therapeutically beneficial in cancer.
autophagy; fasting; hypoglycemia; cancer; oncocytoma
In this issue of Cancer Discovery, Geng and colleagues report on their use of a combination of promoter cytosine methylation profiling with gene expression and ChIP sequencing to elucidate molecular signatures of adult B-acute lymphoblastic leukemia patient samples with BCR–ABL1, E2A–PBX1, and MLL rearrangements. The unique epigenetic and gene expression signatures of these clinically unfavorable B-ALL subtypes identify novel biomarkers and provide a strong rationale for repurposing existing therapies to treat these molecularly distinct diseases.
In this issue of Cancer Discovery, Guagnano and colleagues utilise a
large and diverse annotated collection of cancer cell lines, the “Cancer
Cell Line Encyclopedia”, to correlate whole genome expression and
genomic alteration datasets with cell line sensitivity data to the novel
pan-FGFR inhibitor NVP-BGJ398. Their findings not only underscore the
preclinical utility of such cell line panels in identifying predictive
biomarkers, but also the emergence of the FGFRs as valid therapeutic targets,
across an increasingly broad range of malignancies.
Ingle and colleagues present timely findings identifying genetic variants
associated with response to selective estrogen receptor modulator therapy that
when substantiated in follow-up may represent an important step towards
understanding estrogen-dependent induction of BRCA1 expression and advancing
individualized preventive medicine in women at high risk for developing breast
Breast cancer mortality is principally due to tumor recurrence; however, the molecular mechanisms underlying this process are poorly understood. We now demonstrate that the suppressor of cytokine signaling protein SPSB1 is spontaneously upregulated during mammary tumor recurrence and is both necessary and sufficient to promote tumor recurrence in genetically engineered mouse models. The recurrence-promoting effects of SPSB1 result from its ability to protect cells from apoptosis induced by HER2/neu pathway inhibition or chemotherapy. This, in turn, is attributable to SPSB1 potentiation of c-MET signaling, such that preexisting SPSB1-overexpressing tumor cells are selected for following HER2/neu downregulation. Consistent with this, SPSB1 expression is positively correlated with c-MET activity in human breast cancers and with an increased risk of relapse in patients with breast cancer in a manner that is dependent upon c-MET activity. Our findings define a novel pathway that contributes to breast cancer recurrence and provide the first evidence implicating SPSB proteins in cancer.
The principal cause of death from breast cancer is recurrence. This study identifies SPSB1 as a critical mediator of breast cancer recurrence, suggests activation of the SPSB1/c-MET pathway as an important mechanism of therapeutic resistance in breast cancers, and emphasizes that pharmacological targets for recurrence may be unique to this stage of tumor progression.
SPSB1; c-MET; breast cancer; recurrence; apoptosis
Most melanomas harbor oncogenic BRAFV600 mutations, which constitutively activate the MAP kinase (MAPK) pathway. Although MAPK pathway inhibitors show clinical benefit in BRAFV600-mutant melanoma, it remains incompletely understood why 10-20% of patients fail to respond. Here, we show that RAF inhibitor sensitive and resistant BRAFV600-mutant melanomas display distinct transcriptional profiles. Whereas most drug-sensitive cell lines and patient biopsies showed high expression and activity of the melanocytic lineage transcription factor MITF, intrinsically resistant cell lines and biopsies displayed low MITF expression but higher levels of NF-κB signaling and the receptor tyrosine kinase AXL. In vitro, these MITF-low/NF-κB-high melanomas were resistant to inhibition of RAF and MEK, singly or in combination, and ERK. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF or NF-κB activity may influence intrinsic resistance to MAPK pathway inhibitors in BRAFV600-mutant melanoma.
MITF; NF-κB; MAPK pathway; melanoma; intrinsic resistance
Despite its clinical importance, very little is known about the natural history and molecular underpinnings of lung cancer dissemination and metastasis. Here we employed a genetically-engineered mouse model of metastatic lung adenocarcinoma in which cancer cells are fluorescently marked to determine whether dissemination is an inherent ability or a major acquired phenotype during lung adenocarcinoma metastasis. We find very little evidence for dissemination from oncogenic Kras-driven hyperplasias or most adenocarcinomas. p53 loss is insufficient to drive dissemination but rather enables rare cancer cells in a small fraction of primary adenocarcinomas to gain alterations that drive dissemination. Molecular characterization of disseminated tumors cells indicates that down-regulation of the transcription factor Nkx2-1 precedes dissemination. Finally, we show that metastatic primary tumors possess a highly proliferative sub-population of cells with characteristics matching those of disseminating cells. We propose that dissemination is a major hurdle during the natural course of lung adenocarcinoma metastasis.
Metastasis; genetically-engineered mouse model; lung cancer; dissemination; Nkx2-1
Metabolomic analyses of human tumors and mouse models of cancer have identified key roles for autophagy in supporting mitochondrial metabolism and homeostasis. In this review, we highlight data suggesting that autophagy inhibition may be particularly effective in Braf-driven malignancies. Catalytic BRAF inhibitors have profound efficacy in tumors carrying activating mutations in Braf but are limited by the rapid emergence of resistance due in part to increased mitochondrial biogenesis and heightened rates of oxidative phosphorylation. We suggest that combined inhibition of autophagy and BRAF may overcome this limitation.
BrafV600E-driven tumors require autophagy and likely autophagy-provided substrates to maintain mitochondrial metabolism and to promote tumor growth, suggesting that autophagy ablation may improve cancer therapy.
Autophagy; metabolism; oncocytoma; mitochondria; glutamine; BRAF
Autophagy inhibition is a potential therapeutic strategy in cancer, but it is unknown which tumors will benefit. The BRAFV600E mutation has been identified as important in pediatric CNS tumors and is known to affect autophagy in other tumor types. We evaluated CNS tumor cells with BRAFV600E and found that mutant cells (but not wild type) display high rates of induced autophagy, are sensitive to pharmacologic and genetic autophagy inhibition, and display synergy when the clinically used autophagy inhibitor chloroquine was combined with the Raf inhibitor vemurafenib or standard chemotherapeutics. Importantly we also demonstrate chloroquine can improve vemurafenib sensitivity in a resistant ex vivo primary culture and provide the first demonstration in a patient harboring the V600E mutation treated with vemurafenib that addition of chloroquine can improve clinical outcomes. These findings suggest CNS tumors with BRAFV600E are autophagy-dependent and should be targeted with autophagy inhibition in combination with other therapeutic strategies.
Brain tumors; pediatric; autophagy; BRAF; chloroquine
Disease recurrence is the most common cause of death for breast cancer patients, yet little is known about the molecular mechanisms underlying this process. Using inducible transgenic mouse model systems, Feng and colleagues identified SPSB1 as a determinant of breast cancer recurrence by virtue of its ability to protect tumor cells from apoptosis through c-MET activation.
Recently identified IDH mutations lead to the production of 2-hydroxyglutarate (2HG), an onco-metabolite aberrantly elevated in selected cancers. We developed a facile and inexpensive fluorimetric microplate assay for quantitation of 2HG and performed an unbiased small molecule screen in live cells to identify compounds capable of perturbing 2HG production. Zaprinast, a PDE5 inhibitor, was identified as an efficacious modulator of 2HG production and confirmed to lower 2HG levels in vivo. The mechanism of action was not due to cGMP stabilization, but rather, profiling of metabolites upstream of mutant IDH1 pointed to targeted inhibition of the enzyme glutaminase (GLS). Zaprinast treatment reversed histone hypermethylation and soft agar growth of IDH1 mutant cells, and treatment of glutamine-addicted pancreatic cancer cells reduced growth and sensitized cells to oxidative damage. Thus, Zaprinast is efficacious against glutamine metabolism and further establishes the therapeutic linkages between GLS and 2HG-mediated oncogenesis.
Approximately half of the familial aggregation of breast cancer remains unexplained. A multiple-case breast cancer family exome sequencing study identified three likely pathogenic mutations in RINT1 (NM_021930.4) not present in public sequencing databases: RINT1 c.343C>T (p.Q115X), c.1132_1134del (p.M378del) and c.1207G>T (p.D403Y). Based on this finding, a population-based case-control mutation-screening study was conducted and identified 29 carriers of rare (MAF < 0.5%), likely pathogenic variants: 23 in 1,313 early-onset breast cancer cases and 6 in 1,123 frequency-matched controls (OR=3.24, 95%CI 1.29-8.17; p=0.013). RINT1 mutation screening of probands from 798 multiple-case breast cancer families identified 4additional carriers of rare genetic variants. Analysis of the incidence of first primary cancers in families of women in RINT1-mutation carrying families estimated that carriers were at increased risks of Lynch syndrome-spectrum cancers (SIR 3.35, 95% CI 1.7-6.0; P=0.005), particularly for relatives diagnosed with cancer under age 60 years (SIR 10.9, 95%CI 4.7-21; P=0.0003).
Breast Cancer; RINT1; Lynch Syndrome; Genetic Susceptibility
Neoplastic cells rely on the tumor microenvironment (TME) for survival and progression factors. Indeed, senescent and cancer-associated fibroblasts (CAFs) express factors that promote tumorigenesis that are collectively referred to as the senescence-associated secretory phenotype (SASP). Despite their importance in tumorigenesis, the mechanisms that control TME-derived factor expression remain poorly understood. Here we address a key unanswered question, how the SASP is sustained in senescent fibroblasts and CAFs. We find that the mitogen-activated protein kinase p38 (p38MAPK) controls AUF1 occupancy on SASP mRNAs and thus controls their stability. The importance of this regulatory mechanism is underscored by our findings that stromal-specific p38MAPK inhibition abrogates the tumor-promoting activities of CAFs and senescent fibroblasts. Our data suggest that targeting SASP mRNA stability through inhibition of p38MAPK will significantly aid the development of clinical strategies to target the TME.
tumor microenvironment; senescence; SASP; mRNA stability; p38MAPK
Non-small cell lung cancers (NSCLC) harboring anaplastic lymphoma kinase (ALK) gene rearrangements invariably develop resistance to the ALK tyrosine kinase inhibitor (TKI) crizotinib. Herein, we report the first preclinical evaluation of the next-generation ALK TKI, ceritinib (LDK378) in the setting of crizotinib resistance. Interrogation of in vitro and in vivo models of acquired resistance to crizotinib, including cell lines established from biopsies of crizotinib-resistant NSCLC patients revealed that ceritinib potently overcomes crizotinib resistance mutations. In particular, ceritinib effectively inhibits ALK harboring L1196M, G1269A, I1171T and S1206Y mutations, and a co-crystal of ceritinib bound to ALK provides structural bases for this increased potency. However, we observed that ceritinib did not overcome two crizotinib-resistant ALK mutations, G1202R and F1174C, and one of these mutations were identified in 5 out of 11 biopsies from patients with acquired resistance to ceritinib. Altogether our results demonstrate that ceritinib can overcome crizotinib resistance, consistent with clinical data showing marked efficacy of ceritinib in patients with crizotinib-resistant disease.
NSCLC; ALK; ceritinib; LDK378; resistance mechanisms