Stromal cells residing in the tumor microenvironment contribute to the development of therapy resistance. Here we show that chemotherapy-educated mesenchymal stem cells (MSC) promote therapy resistance via cross-talk with tumor-initiating cells (TIC), a resistant tumor cell subset that initiates tumorigenesis and metastasis. In response to gemcitabine chemotherapy, MSCs colonized pancreatic adenocarcinomas in large numbers and resided in close proximity to TICs. Furthermore, gemcitabine-educated MSCs promoted the enrichment of TICs in vitro and enhance tumor growth in vivo. These effects were dependent on the secretion of CXCL10 by gemcitabine-educated MSCs and subsequent activation of the CXCL10–CXCR3 axis in TICs. In an orthotopic pancreatic tumor model, targeting TICs using nanovesicles (called nanoghosts) derived from MSC membranes and loaded with a CXCR3 antagonist enhanced therapy outcome and delayed tumor regrowth when administered in combination with gemcitabine. Overall, our results establish a mechanism through which MSCs promote chemoresistance, and propose a novel drug delivery system to target TICs and overcome this resistance.Significance: These results establish a mechanism by which mesenchyme stem cells in the tumor microenvironment promote chemoresistance, and they propose a novel drug delivery system to overcome this challenge. Cancer Res; 78(5); 1253–65. ©2018 AACR.
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Cancer Medicine by Alexandros G.Sfakianakis,Anapafseos 5 Agios Nikolaos,Crete 72100,Greece,tel :00302841026182 & 00306932607174
Τετάρτη 28 Φεβρουαρίου 2018
Therapy-Educated Mesenchymal Stem Cells Enrich for Tumor-Initiating Cells
A KDM5 Inhibitor Increases Global H3K4 Trimethylation Occupancy and Enhances the Biological Efficacy of 5-Aza-2'-Deoxycytidine
The H3K4 demethylase KDM5B is amplified and overexpressed in luminal breast cancer, suggesting it might constitute a potential cancer therapy target. Here, we characterize, in breast cancer cells, the molecular effects of a recently developed small-molecule inhibitor of the KDM5 family of proteins (KDM5i), either alone or in combination with the DNA-demethylating agent 5-aza-2′-deoxycytidine (DAC). KDM5i treatment alone increased expression of a small number of genes, whereas combined treatment with DAC enhanced the effects of the latter for increasing expression of hundreds of DAC-responsive genes. ChIP-seq studies revealed that KDM5i resulted in the broadening of existing H3K4me3 peaks. Furthermore, cells treated with the drug combination exhibited increased promoter and gene body H3K4me3 occupancy at DAC-responsive genes compared with DAC alone. Importantly, treatment with either DAC or DAC+KDM5i induced a dramatic increase in H3K27ac at enhancers with an associated significant increase in target gene expression, suggesting a previously unappreciated effect of DAC on transcriptional regulation. KDM5i synergized with DAC to reduce the viability of luminal breast cancer cells in in vitro assays. Our study provides the first look into the molecular effects of a novel KDM5i compound and suggests that combinatorial inhibition along with DAC represents a new area to explore in translational epigenetics.Significance: This study offers a first look into the molecular effects of a novel KDM5 inhibitory compound, suggesting how its use in combination with DNA methylation inhibitors presents new opportunities to explore in translational cancer epigenetics. Cancer Res; 78(5); 1127–39. ©2017 AACR.
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Race Disparities in the Contribution of miRNA Isoforms and tRNA-Derived Fragments to Triple-Negative Breast Cancer
Triple-negative breast cancer (TNBC) is a breast cancer subtype characterized by marked differences between White and Black/African-American women. We performed a systems-level analysis on datasets from The Cancer Genome Atlas to elucidate how the expression patterns of mRNAs are shaped by regulatory noncoding RNAs (ncRNA). Specifically, we studied isomiRs, that is, isoforms of miRNAs, and tRNA-derived fragments (tRF). In normal breast tissue, we observed a marked cohesiveness in both the ncRNA and mRNA layers and the associations between them. This cohesiveness was widely disrupted in TNBC. Many mRNAs become either differentially expressed or differentially wired between normal breast and TNBC in tandem with isomiR or tRF dysregulation. The affected pathways included energy metabolism, cell signaling, and immune responses. Within TNBC, the wiring of the affected pathways with isomiRs and tRFs differed in each race. Multiple isomiRs and tRFs arising from specific miRNA loci (e.g., miR-200c, miR-21, the miR-17/92 cluster, the miR-183/96/182 cluster) and from specific tRNA loci (e.g., the nuclear tRNAGly and tRNALeu, the mitochondrial tRNAVal and tRNAPro) were strongly associated with the observed race disparities in TNBC. We highlight the race-specific aspects of transcriptome wiring by discussing in detail the metastasis-related MAPK and the Wnt/β-catenin signaling pathways, two of the many key pathways that were found differentially wired. In conclusion, by employing a data- and knowledge-driven approach, we comprehensively analyzed the normal and cancer transcriptomes to uncover novel key contributors to the race-based disparities of TNBC.Significance: This big data-driven study comparing normal and cancer transcriptomes uncovers RNA expression differences between Caucasian and African-American patients with triple-negative breast cancer that might help explain disparities in incidence and aggressive character. Cancer Res; 78(5); 1140–54. ©2017 AACR.
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Loss of RASSF4 Expression in Multiple Myeloma Promotes RAS-Driven Malignant Progression
RAS mutations occur frequently in multiple myeloma (MM), but apart from driving progression, they can also stimulate antitumor effects by activating tumor-suppressive RASSF proteins. Although this family of death effector molecules are often silenced in cancers, functional data about RASSF proteins in MM are lacking. Here, we report that RASSF4 is downregulated during MM progression and correlates with a poor prognosis. Promoter methylation analysis in human cell lines revealed an inverse correlation between RASSF4 mRNA levels and methylation status. Epigenetic modulating agents restored RASSF4 expression. Enforced expression of RASSF4 induced G2-phase cell-cycle arrest and apoptosis in human cell lines, reduced primary MM cell viability, and blocked MM growth in vivo. Mechanistic investigations showed that RASSF4 linked RAS to several pro-death pathways, including those regulated by the kinases MST1, JNK, and p38. By activating MST1 and the JNK/c-Jun pathway, RASSF4 sensitized MM cells to bortezomib. Genetic or pharmacological elevation of RASSF4 levels increased the anti-MM effects of the clinical relevant MEK1/2 inhibitor trametinib. Kinome analysis revealed that this effect was mediated by concomitant activation of the JNK/c-Jun pathway along with inactivation of the MEK/ERK and PI3K/mTOR/Akt pathways. Overall, our findings establish RASSF4 as a tumor-suppressive hub in MM and provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib to treat patients with tumors exhibiting low RASSF4 expression.Significance: These findings provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib in patients with multiple myeloma whose tumors exhibit low RASSF4 expression. Cancer Res; 78(5); 1155–68. ©2017 AACR.
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Combined Mutation of Apc, Kras, and Tgfbr2 Effectively Drives Metastasis of Intestinal Cancer
Colorectal cancer is driven by the accumulation of driver mutations, but the contributions of specific mutations to different steps in malignant progression are not fully understood. In this study, we generated mouse models harboring different combinations of key colorectal cancer driver mutations (Apc, Kras, Tgfbr2, Trp53, Fbxw7) in intestinal epithelial cells to comprehensively investigate their roles in the development of primary tumors and metastases. ApcΔ716 mutation caused intestinal adenomas and combination with Trp53R270H mutation or Tgfbr2 deletion induced submucosal invasion. The addition of KrasG12D mutation yielded epithelial–mesenchymal transition (EMT)-like morphology and lymph vessel intravasation of the invasive tumors. In contrast, combinations of ApcΔ716 with KrasG12D and Fbxw7 mutation were insufficient for submucosal invasion, but still induced EMT-like histology. Studies using tumor-derived organoids showed that KrasG12D was critical for liver metastasis following splenic transplantation, when this mutation was combined with either ApcΔ716 plus Trp53R270H or Tgfbr2 deletion, with the highest incidence of metastasis displayed by tumors with a ApcΔ716 KrasG12D Tgfbr2−/− genotype. RNA sequencing analysis of tumor organoids defined distinct gene expression profiles characteristic for the respective combinations of driver mutations, with upregulated genes in ApcΔ716 KrasG12D Tgfbr2−/− tumors found to be similarly upregulated in specimens of human metastatic colorectal cancer. Our results show how activation of Wnt and Kras with suppression of TGFβ signaling in intestinal epithelial cells is sufficient for colorectal cancer metastasis, with possible implications for the development of metastasis prevention strategies.Significance: These findings illuminate how key driver mutations in colon cancer cooperate to drive the development of metastatic disease, with potential implications for the development of suitable prevention strategies. Cancer Res; 78(5); 1334–46. ©2017 AACR.
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Long Noncoding RNA pancEts-1 Promotes Neuroblastoma Progression through hnRNPK-Mediated {beta}-Catenin Stabilization
Long noncoding RNAs (lncRNA) play essential roles in tumor progression. However, the functions of lncRNAs in the tumorigenesis and aggressiveness of neuroblastoma still remain to be determined. Here, we report the identification of lncRNA pancEts-1 as a novel driver of neuroblastoma progression by using a public microarray dataset. LncRNA pancEts-1 promoted the growth, invasion, and metastasis of neuroblastoma cells in vitro and in vivo. Mechanistically, pancEts-1 bound to hnRNPK to facilitate its physical interaction with β-catenin, whereas hnRNPK stabilized the β-catenin by inhibiting proteasome-mediated degradation, resulting in transcriptional alteration of target genes associated with neuroblastoma progression. Both pancEts-1 and hnRNPK were upregulated in clinical neuroblastoma tissues, and were associated with unfavorable outcome of patients. Overall, our results define an oncogenic role of pancEts-1 in neuroblastoma progression through hnRNPK-mediated β-catenin stabilization, with potential implications for the clinical therapeutics of neuroblastoma.Significance: These findings reveal the oncogenic functions of a long noncoding RNA in neuroblastoma progression, offering a potential target for clinical therapeutics. Cancer Res; 78(5); 1169–83. ©2018 AACR.
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FIH Is an Oxygen Sensor in Ovarian Cancer for G9a/GLP-Driven Epigenetic Regulation of Metastasis-Related Genes
The prolyl hydroxylase domain-containing proteins (PHD1-3) and the asparaginyl hydroxlyase factor inhibiting HIF (FIH) are oxygen sensors for hypoxia-inducible factor-driven transcription of hypoxia-induced genes, but whether these sensors affect oxygen-dependent epigenetic regulation more broadly is not known. Here, we show that FIH exerts an additional role as an oxygen sensor in epigenetic control by the histone lysine methyltransferases G9a and GLP. FIH hydroxylated and inhibited G9a and GLP under normoxia. When the FIH reaction was limited under hypoxia, G9a and GLP were activated and repressed metastasis suppressor genes, thereby triggering cancer cell migration and peritoneal dissemination of ovarian cancer xenografts. In clinical specimens of ovarian cancer, expression of FIH and G9a were reciprocally associated with patient outcomes. We also identified mutations of FIH target motifs in G9a and GLP, which exhibited excessive H3K9 methylation and facilitated cell invasion. This study provides insight into a new function of FIH as an upstream regulator of oxygen-dependent chromatin remodeling. It also implies that the FIH-G9a/GLP pathway could be a potential target for inhibiting hypoxia-induced cancer metastasis.Significance: These findings deepen understanding of oxygen-dependent gene regulation and cancer metastasis in response to hypoxia. Cancer Res; 78(5); 1184–99. ©2017 AACR.
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Deletion of the von Hippel-Lindau Gene in Hemangioblasts Causes Hemangioblastoma-like Lesions in Murine Retina
von Hippel-Lindau (VHL) disease is an autosomal-dominant tumor predisposition syndrome characterized by the development of highly vascularized tumors and cysts. LOH of the VHL gene results in aberrant upregulation of hypoxia-inducible factors (HIF) and has been associated with tumor formation. Hemangioblastomas of the central nervous system and retina represent the most prevalent VHL-associated tumors, but no VHL animal model has reproduced retinal capillary hemangioblastomas (RCH), the hallmark lesion of ocular VHL. Here we report our work in developing a murine model of VHL-associated RCH by conditionally inactivating Vhl in a hemangioblast population using a Scl-Cre-ERT2 transgenic mouse line. In transgenic mice carrying the conditional allele and the Scl-Cre-ERT2 allele, 64% exhibited various retinal vascular anomalies following tamoxifen induction. Affected Vhl-mutant mice demonstrated retinal vascular lesions associated with prominent vasculature, anomalous capillary networks, hemorrhage, exudates, and localized fibrosis. Histologic analyses showed RCH-like lesions characterized by tortuous, dilated vasculature surrounded by "tumorlet" cell cluster and isolated foamy stromal cells, which are typically associated with RCH. Fluorescein angiography suggested increased vascular permeability of the irregular retinal vasculature and hemangioblastoma-like lesions. Vhl deletion was detected in "tumorlet" cells via microdissection. Our findings provide a phenotypic recapitulation of VHL-associated RCH in a murine model that may be useful to study RCH pathogenesis and therapeutics aimed at treating ocular VHL.Significance: This study describes a model that phenotypically recapitulates a form of retinal pathogenesis that is driven by genetic loss of the VHL tumor suppressor, providing a useful tool for its study and therapeutic intervention. Cancer Res; 78(5); 1266–74. ©2018 AACR.
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Familial and Somatic BAP1 Mutations Inactivate ASXL1/2-Mediated Allosteric Regulation of BAP1 Deubiquitinase by Targeting Multiple Independent Domains
Deleterious mutations of the ubiquitin carboxy-terminal hydrolase BAP1 found in cancers are predicted to encode inactive truncated proteins, suggesting that loss of enzyme function is a primary tumorigenic mechanism. However, many tumors exhibit missense mutations or in-frame deletions or insertions, often outside the functionally critical UCH domain in this tumor suppressor protein. Thus, precisely how these mutations inactivate BAP1 is unknown. Here, we show how these mutations affect BAP1 interactions with the Polycomb group-like protein, ASXL2, using combinations of computational modeling technology, molecular biology, and in vitro reconstitution biochemistry. We found that the BAP1–ASXL2 interaction is direct and high affinity, occurring through the ASXH domain of ASXL2, an obligate partner for BAP1 enzymatic activity. The ASXH domain was the minimal domain for binding the BAP1 ULD domain, and mutations on the surfaces of predicted helices of ASXH abolished BAP1 association and stimulation of BAP1 enzymatic activity. The BAP1-UCH, BAP1-ULD, and ASXH domains formed a cooperative stable ternary complex required for deubiquitination. We defined four classes of alterations in BAP1 outside the UCH domain, each failing to productively recruit ASXH to the wild-type BAP1 catalytic site via the ULD, resulting in loss of BAP1 ubiquitin hydrolase activity. Our results indicate that many BAP1 mutations act allosterically to inhibit ASXH binding, thereby leading to loss of enzyme activity. Small-molecule approaches to reactivate latent wild-type UCH activity of these mutants might be therapeutically viable.Significance: Combined computational and biochemical approaches demonstrate that the BAP1–ASXL2 interaction is direct and high affinity and that many BAP1 mutations act allosterically to inhibit BAP1–ASXL2 binding. Cancer Res; 78(5); 1200–13. ©2017 AACR.
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Inactivation of Cancer-Associated-Fibroblasts Disrupts Oncogenic Signaling in Pancreatic Cancer Cells and Promotes Its Regression
Resident fibroblasts that contact tumor epithelial cells (TEC) can become irreversibly activated as cancer-associated-fibroblasts (CAF) that stimulate oncogenic signaling in TEC. In this study, we evaluated the cross-talk between CAF and TEC isolated from tumors generated in a mouse model of KRAS/mut p53-induced pancreatic cancer (KPC mice). Transcriptomic profiling conducted after treatment with the anticancer compound Minnelide revealed deregulation of the TGFβ signaling pathway in CAF, resulting in an apparent reversal of their activated state to a quiescent, nonproliferative state. TEC exposed to media conditioned by drug-treated CAFs exhibited a decrease in oncogenic signaling, as manifested by downregulation of the transcription factor Sp1. This inhibition was rescued by treating TEC with TGFβ. Given promising early clinical studies with Minnelide, our findings suggest that approaches to inactivate CAF and prevent tumor–stroma cross-talk may offer a viable strategy to treat pancreatic cancer.Significance: In an established mouse model of pancreatic cancer, administration of the promising experimental drug Minnelide was found to actively deplete reactive stromal fibroblasts and to trigger tumor regression, with implications for stromal-based strategies to attack this disease. Cancer Res; 78(5); 1321–33. ©2018 AACR.
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The CARMA3-Bcl10-MALT1 Signalosome Drives NF{kappa}B Activation and Promotes Aggressiveness in Angiotensin II Receptor-Positive Breast Cancer
The angiotensin II receptor AGTR1, which mediates vasoconstrictive and inflammatory signaling in vascular disease, is overexpressed aberrantly in some breast cancers. In this study, we established the significance of an AGTR1-responsive NFκB signaling pathway in this breast cancer subset. We documented that AGTR1 overexpression occurred in the luminal A and B subtypes of breast cancer, was mutually exclusive of HER2 expression, and correlated with aggressive features that include increased lymph node metastasis, reduced responsiveness to neoadjuvant therapy, and reduced overall survival. Mechanistically, AGTR1 overexpression directed both ligand-independent and ligand-dependent activation of NFκB, mediated by a signaling pathway that requires the triad of CARMA3, Bcl10, and MALT1 (CBM signalosome). Activation of this pathway drove cancer cell–intrinsic responses that include proliferation, migration, and invasion. In addition, CBM-dependent activation of NFκB elicited cancer cell–extrinsic effects, impacting endothelial cells of the tumor microenvironment to promote tumor angiogenesis. CBM/NFκB signaling in AGTR1+ breast cancer therefore conspires to promote aggressive behavior through pleiotropic effects. Overall, our results point to the prognostic and therapeutic value of identifying AGTR1 overexpression in a subset of HER2-negative breast cancers, and they provide a mechanistic rationale to explore the repurposing of drugs that target angiotensin II–dependent NFκB signaling pathways to improve the treatment of this breast cancer subset.Significance: These findings offer a mechanistic rationale to explore the repurposing of drugs that target angiotensin action to improve the treatment of AGTR1-expressing breast cancers. Cancer Res; 78(5); 1225–40. ©2017 AACR.
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Statin-Induced Cancer Cell Death Can Be Mechanistically Uncoupled from Prenylation of RAS Family Proteins
The statin family of drugs preferentially triggers tumor cell apoptosis by depleting mevalonate pathway metabolites farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which are used for protein prenylation, including the oncoproteins of the RAS superfamily. However, accumulating data indicate that activation of the RAS superfamily are poor biomarkers of statin sensitivity, and the mechanism of statin-induced tumor-specific apoptosis remains unclear. Here we demonstrate that cancer cell death triggered by statins can be uncoupled from prenylation of the RAS superfamily of oncoproteins. Ectopic expression of different members of the RAS superfamily did not uniformly sensitize cells to fluvastatin, indicating that increased cellular demand for protein prenylation cannot explain increased statin sensitivity. Although ectopic expression of HRAS increased statin sensitivity, expression of myristoylated HRAS did not rescue this effect. HRAS-induced epithelial-to-mesenchymal transition (EMT) through activation of zinc finger E-box binding homeobox 1 (ZEB1) sensitized tumor cells to the antiproliferative activity of statins, and induction of EMT by ZEB1 was sufficient to phenocopy the increase in fluvastatin sensitivity; knocking out ZEB1 reversed this effect. Publicly available gene expression and statin sensitivity data indicated that enrichment of EMT features was associated with increased sensitivity to statins in a large panel of cancer cell lines across multiple cancer types. These results indicate that the anticancer effect of statins is independent from prenylation of RAS family proteins and is associated with a cancer cell EMT phenotype.Significance: The use of statins to target cancer cell EMT may be useful as a therapy to block cancer progression. Cancer Res; 78(5); 1347–57. ©2017 AACR.
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Nkx2.8 Inhibits Epithelial-Mesenchymal Transition in Bladder Urothelial Carcinoma via Transcriptional Repression of Twist1
Epithelial-to-mesenchymal transition (EMT) promotes metastasis, which is the main cause of bladder urothelial carcinoma–related death. Loss of the candidate tumor-suppressor gene Nkx2.8 has been associated with urothelial carcinoma lymph node metastasis. Here, we show that enforced expression of Nkx2.8 is sufficient to inhibit EMT, reduce motility, and blunt invasiveness of urothelial carcinoma cells. Mechanistic investigations showed that Nkx2.8 negatively regulated expression of the EMT inducer Twist1 in urothelial carcinoma cells, at both the level of mRNA and protein accumulation. Nkx2.8 bound directly to the promoter region of this gene and transcriptionally repressed its expression. Twist1 upregulation reversed EMT inhibition by Nkx2.8, restoring the invasive phenotype of urothelial carcinoma cells. In clinical urothelial carcinoma specimens, expression of Nkx2.8 inversely correlated with Twist1 expression, and urothelial carcinoma patients with Nkx2.8 positivity and low Twist1 expression displayed the best prognosis. Our findings highlight the Nkx2.8–Twist1 axis as candidate target for therapeutic intervention in advanced urothelial carcinoma.Significance: These findings highlight a novel EMT signaling axis as a candidate target for therapeutic intervention in advanced urothelial carcinomas. Cancer Res; 78(5); 1241–52. ©2018 AACR.
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Brain-Mimetic 3D Culture Platforms Allow Investigation of Cooperative Effects of Extracellular Matrix Features on Therapeutic Resistance in Glioblastoma
Glioblastoma (GBM) tumors exhibit potentially actionable genetic alterations against which targeted therapies have been effective in treatment of other cancers. However, these therapies have largely failed in GBM patients. A notable example is kinase inhibitors of EGFR, which display poor clinical efficacy despite overexpression and/or mutation of EGFR in >50% of GBM. In addressing this issue, preclinical models may be limited by the inability to accurately replicate pathophysiologic interactions of GBM cells with unique aspects of the brain extracellular matrix (ECM), which is relatively enriched in hyaluronic acid (HA) and flexible. In this study, we present a brain-mimetic biomaterial ECM platform for 3D culturing of patient-derived GBM cells, with improved pathophysiologic properties as an experimental model. Compared with orthotopic xenograft assays, the novel biomaterial cultures we developed better preserved the physiology and kinetics of acquired resistance to the EGFR inhibition than gliomasphere cultures. Orthogonal modulation of both HA content and mechanical properties of biomaterial scaffolds was required to achieve this result. Overall, our findings show how specific interactions between GBM cell receptors and scaffold components contribute significantly to resistance to the cytotoxic effects of EGFR inhibition.Significance: Three-dimensional culture scaffolds of glioblastoma provide a better physiological representation over current methods of patient-derived cell culture and xenograft models. Cancer Res; 78(5); 1358–70. ©2017 AACR.
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Molecular Pharmacodynamics-Guided Scheduling of Biologically Effective Doses: A Drug Development Paradigm Applied to MET Tyrosine Kinase Inhibitors
The development of molecularly targeted agents has benefited from use of pharmacodynamic markers to identify "biologically effective doses" (BED) below MTDs, yet this knowledge remains underutilized in selecting dosage regimens and in comparing the effectiveness of targeted agents within a class. We sought to establish preclinical proof-of-concept for such pharmacodynamics-based BED regimens and effectiveness comparisons using MET kinase small-molecule inhibitors. Utilizing pharmacodynamic biomarker measurements of MET signaling (tumor pY1234/1235MET/total MET ratio) in a phase 0–like preclinical setting, we developed optimal dosage regimens for several MET kinase inhibitors and compared their antitumor efficacy in a MET-amplified gastric cancer xenograft model (SNU-5). Reductions in tumor pY1234/1235MET/total MET of 95%–99% were achievable with tolerable doses of EMD1214063/MSC2156119J (tepotinib), XL184 (cabozantinib), and XL880/GSK1363089 (foretinib), but not ARQ197 (tivantinib), which did not alter the pharmacodynamic biomarker. Duration of kinase suppression and rate of kinase recovery were specific to each agent, emphasizing the importance of developing customized dosage regimens to achieve continuous suppression of the pharmacodynamic biomarker at the required level (here, ≥90% MET kinase suppression). The customized dosage regimen of each inhibitor yielded substantial and sustained tumor regression; the equivalent effectiveness of customized dosage regimens that achieve the same level of continuous molecular target control represents preclinical proof-of-concept and illustrates the importance of proper scheduling of targeted agent BEDs. Pharmacodynamics-guided biologically effective dosage regimens (PD-BEDR) potentially offer a superior alternative to pharmacokinetic guidance (e.g., drug concentrations in surrogate tissues) for developing and making head-to-head comparisons of targeted agents. Mol Cancer Ther; 17(3); 698–709. ©2018 AACR.
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Inhibition of FLT3 and PIM Kinases by EC-70124 Exerts Potent Activity in Preclinical Models of Acute Myeloid Leukemia
Internal tandem duplication (ITD) or tyrosine kinase domain mutations of FLT3 is the most frequent genetic alteration in acute myelogenous leukemia (AML) and are associated with poor disease outcome. Despite considerable efforts to develop single-target FLT3 drugs, so far, the most promising clinical response has been achieved using the multikinase inhibitor midostaurin. Here, we explore the activity of the indolocarbazole EC-70124, from the same chemical space as midostaurin, in preclinical models of AML, focusing on those bearing FLT3-ITD mutations. EC-70124 potently inhibits wild-type and mutant FLT3, and also other important kinases such as PIM kinases. EC-70124 inhibits proliferation of AML cell lines, inducing cell-cycle arrest and apoptosis. EC-70124 is orally bioavailable and displays higher metabolic stability and lower human protein plasma binding compared with midostaurin. Both in vitro and in vivo pharmacodynamic analyses demonstrate inhibition of FLT3-STAT5, Akt-mTOR-S6, and PIM-BAD pathways. Oral administration of EC-70124 in FLT3-ITD xenograft models demonstrates high efficacy, reaching complete tumor regression. Ex vivo, EC-70124 impaired cell viability in leukemic blasts, especially from FLT3-ITD patients. Our results demonstrate the ability of EC-70124 to reduce proliferation and induce cell death in AML cell lines, patient-derived leukemic blast and xenograft animal models, reaching best results in FLT3 mutants that carry other molecular pathways' alterations. Thus, its unique inhibition profile warrants EC-70124 as a promising agent for AML treatment based on its ability to interfere the complex oncogenic events activated in AML at several levels. Mol Cancer Ther; 17(3); 614–24. ©2018 AACR.
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A DNA-Interacting Payload Designed to Eliminate Cross-Linking Improves the Therapeutic Index of Antibody-Drug Conjugates (ADCs)
Tumor-selective delivery of cytotoxic agents in the form of antibody–drug conjugates (ADCs) is now a clinically validated approach for cancer treatment. In an attempt to improve the clinical success rate of ADCs, emphasis has been recently placed on the use of DNA–cross-linking pyrrolobenzodiazepine compounds as the payload. Despite promising early clinical results with this class of ADCs, doses achievable have been low due to systemic toxicity. Here, we describe the development of a new class of potent DNA-interacting agents wherein changing the mechanism of action from a cross-linker to a DNA alkylator improves the tolerability of the ADC. ADCs containing the DNA alkylator displayed similar in vitro potency, but improved bystander killing and in vivo efficacy, compared with those of the cross-linker. Thus, the improved in vivo tolerability and antitumor activity achieved in rodent models with ADCs of the novel DNA alkylator could provide an efficacious, yet safer option for cancer treatment. Mol Cancer Ther; 17(3); 650–60. ©2018 AACR.
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A Correlative Analysis of PD-L1, PD-1, PD-L2, EGFR, HER2, and HER3 Expression in Oropharyngeal Squamous Cell Carcinoma
We explored potential associations of the PD-1/PD-L1/PD-L2 pathway with clinical characteristics, outcome, and expression of EGFR, HER2, HER3 in oropharyngeal squamous cell carcinoma (OPSCC) using an institutional database. Protein expression was assessed by IHC on tissue microarray sections (EGFR, HER2, HER3) or whole tissue sections (PD-1/PD-L1/PD-L2). Expression of EGFR, HER2, HER3, PD-L1, and PD-L2 was quantified on tumor cells. Maximum density of PD-1 positive lymphocytes was measured on a scale of 0 to 4 within the tumor mass and peritumoral stroma. Associations between biomarkers and patient outcomes were tested using descriptive and inferential statistics, logistic regression, and Cox proportional hazards models. We analyzed tissue samples from 97 OPSCC cases: median age 59 years, p16+ (71%), male (83.5%), never smokers (18%), stage 3 to 4 disease (77%). Twenty-five percent of cases were PD-L1 positive. The proportion of PD-L1+ tumors was higher in p16+ (29%) than p16– OPSCC (11%, P = 0.047). There was no correlation between PD-L1, PD-L2, PD-1, EGFR, HER2, or HER3 expression. Positive PD-L1 status correlated with advanced nodal disease on multivariate analysis (OR 5.53; 95% CI, 1.06–28.77; P = 0.042). Negative PD-L2 expression was associated with worse survival (HR 3.99; 95% CI, 1.37–11.58; P = 0.011) in p16– OPSCC. Lower density of PD-1 positive lymphocytes in peritumoral stroma was associated with significantly increased risk of death on multivariate analysis (HR 3.17; 95% CI, 1.03–9.78; P = 0.045) after controlling for prognostic factors such as stage and p16 status. PD-L1 expression on tumor cells correlates with p16 status and advanced nodal status in OPSCC. PD-1 positive lymphocytes in peritumoral stroma serve as an independent prognostic factor for overall survival. Mol Cancer Ther; 17(3); 710–6. ©2018 AACR.
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Molecularly Targeted Cancer Combination Therapy with Near-Infrared Photoimmunotherapy and Near-Infrared Photorelease with Duocarmycin-Antibody Conjugate
Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that uses an antibody–photoabsorber conjugate (APC). However, the effect of NIR-PIT can be enhanced when combined with other therapies. NIR photocaging groups, based on the heptamethine cyanine scaffold, have been developed to release bioactive molecules near targets after exposure to light. Here, we investigated the combination of NIR-PIT using panitumumab–IR700 (pan-IR700) and the NIR-releasing compound, CyEt–panitumumab–duocarmycin (CyEt-Pan-Duo). Both pan-IR700 and CyEt-Pan-Duo showed specific binding to the EGFR-expressing MDAMB468 cell line in vitro. In in vivo studies, additional injection of CyEt-Pan-Duo immediately after NIR light exposure resulted in high tumor accumulation and high tumor–background ratio. To evaluate the effects of combination therapy in vivo, tumor-bearing mice were separated into 4 groups: (i) control, (ii NIR-PIT, (iii) NIR-release, (iv) combination of NIR-PIT and NIR-release. Tumor growth was significantly inhibited in all treatment groups compared with the control group (P < 0.05), and significantly prolonged survival was achieved (P < 0.05 vs. control). The greatest therapeutic effect was shown with NIR-PIT and NIR-release combination therapy. In conclusion, combination therapy of NIR-PIT and NIR-release enhanced the therapeutic effects compared with either NIR-PIT or NIR-release therapy alone. Mol Cancer Ther; 17(3); 661–70. ©2017 AACR.
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Histone Methyltransferase EZH2: A Therapeutic Target for Ovarian Cancer
Ovarian cancer is the fifth leading cause of cancer-related deaths in females in the United States. There were an estimated 22,440 new cases and 14,080 deaths due to ovarian cancer in 2017. Most patients present with advanced-stage disease, revealing the urgent need for new therapeutic strategies targeting pathways of tumorigenesis and chemotherapy resistance. While multiple genomic changes contribute to the progression of this aggressive disease, it has become increasingly evident that epigenetic events play a pivotal role in ovarian cancer development. One of the well-studied epigenetic modifiers, the histone methyltransferase EZH2, is a member of polycomb repressive complex 2 (PRC2) and is commonly involved in transcriptional repression. EZH2 is the enzymatic catalytic subunit of the PRC2 complex that can alter gene expression by trimethylating lysine 27 on histone 3 (H3K27). In ovarian cancer, EZH2 is commonly overexpressed and therefore potentially serves as an effective therapeutic target. Multiple small-molecule inhibitors are being developed to target EZH2, which are now in clinical trials. Thus, in this review, we highlight the progress made in EZH2-related research in ovarian cancer and discuss the potential utility of targeting EZH2 with available small-molecule inhibitors for ovarian cancer. Mol Cancer Ther; 17(3); 591–602. ©2018 AACR.
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Molecular Pharmacodynamics-Guided Scheduling of Biologically Effective Doses: A Drug Development Paradigm Applied to MET Tyrosine Kinase Inhibitors
The development of molecularly targeted agents has benefited from use of pharmacodynamic markers to identify "biologically effective doses" (BED) below MTDs, yet this knowledge remains underutilized in selecting dosage regimens and in comparing the effectiveness of targeted agents within a class. We sought to establish preclinical proof-of-concept for such pharmacodynamics-based BED regimens and effectiveness comparisons using MET kinase small-molecule inhibitors. Utilizing pharmacodynamic biomarker measurements of MET signaling (tumor pY1234/1235MET/total MET ratio) in a phase 0–like preclinical setting, we developed optimal dosage regimens for several MET kinase inhibitors and compared their antitumor efficacy in a MET-amplified gastric cancer xenograft model (SNU-5). Reductions in tumor pY1234/1235MET/total MET of 95%–99% were achievable with tolerable doses of EMD1214063/MSC2156119J (tepotinib), XL184 (cabozantinib), and XL880/GSK1363089 (foretinib), but not ARQ197 (tivantinib), which did not alter the pharmacodynamic biomarker. Duration of kinase suppression and rate of kinase recovery were specific to each agent, emphasizing the importance of developing customized dosage regimens to achieve continuous suppression of the pharmacodynamic biomarker at the required level (here, ≥90% MET kinase suppression). The customized dosage regimen of each inhibitor yielded substantial and sustained tumor regression; the equivalent effectiveness of customized dosage regimens that achieve the same level of continuous molecular target control represents preclinical proof-of-concept and illustrates the importance of proper scheduling of targeted agent BEDs. Pharmacodynamics-guided biologically effective dosage regimens (PD-BEDR) potentially offer a superior alternative to pharmacokinetic guidance (e.g., drug concentrations in surrogate tissues) for developing and making head-to-head comparisons of targeted agents. Mol Cancer Ther; 17(3); 698–709. ©2018 AACR.
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Afatinib Is a New Therapeutic Approach in Chordoma with a Unique Ability to Target EGFR and Brachyury
Chordomas are rare bone tumors with no approved therapy. These tumors express several activated tyrosine kinase receptors, which prompted attempts to treat patients with tyrosine kinase inhibitors. Although clinical benefit was observed in phase II clinical trials with imatinib and sorafenib, and sporadically also with EGFR inhibitors, therapies evaluated to date have shown modest activity. With the goal of identifying new drugs with immediate therapeutic potential for chordoma patients, we collected clinically approved drugs and other advanced inhibitors of MET, PDGFRβ, and EGFR tyrosine kinases, and assessed their antiproliferative activity against a panel of chordoma cell lines. Chordoma cell lines were not responsive to MET and PDGFRβ inhibitors. U-CH1 and UM-Chor1 were sensitive to all EGFR inhibitors, whereas the remaining cell lines were generally insensitive to these drugs. Afatinib was the only EGFR inhibitor with activity across the chordoma panel. We then investigated the molecular mechanisms behind the responses observed and found that the antiproliferative IC50s correlate with the unique ability of afatinib to promote degradation of EGFR and brachyury, an embryonic transcription factor considered a key driver of chordoma. Afatinib displayed potent antitumor efficacy in U-CH1, SF8894, CF322, and CF365 chordoma tumor models in vivo. In the panel analyzed, high EGFR phosphorylation and low AXL and STK33 expression correlated with higher sensitivity to afatinib and deserve further investigation as potential biomarkers of response. These data support the use of afatinib in clinical trials and provide the rationale for the upcoming European phase II study on afatinib in advanced chordoma. Mol Cancer Ther; 17(3); 603–13. ©2017 AACR.
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Inhibition of FLT3 and PIM Kinases by EC-70124 Exerts Potent Activity in Preclinical Models of Acute Myeloid Leukemia
Internal tandem duplication (ITD) or tyrosine kinase domain mutations of FLT3 is the most frequent genetic alteration in acute myelogenous leukemia (AML) and are associated with poor disease outcome. Despite considerable efforts to develop single-target FLT3 drugs, so far, the most promising clinical response has been achieved using the multikinase inhibitor midostaurin. Here, we explore the activity of the indolocarbazole EC-70124, from the same chemical space as midostaurin, in preclinical models of AML, focusing on those bearing FLT3-ITD mutations. EC-70124 potently inhibits wild-type and mutant FLT3, and also other important kinases such as PIM kinases. EC-70124 inhibits proliferation of AML cell lines, inducing cell-cycle arrest and apoptosis. EC-70124 is orally bioavailable and displays higher metabolic stability and lower human protein plasma binding compared with midostaurin. Both in vitro and in vivo pharmacodynamic analyses demonstrate inhibition of FLT3-STAT5, Akt-mTOR-S6, and PIM-BAD pathways. Oral administration of EC-70124 in FLT3-ITD xenograft models demonstrates high efficacy, reaching complete tumor regression. Ex vivo, EC-70124 impaired cell viability in leukemic blasts, especially from FLT3-ITD patients. Our results demonstrate the ability of EC-70124 to reduce proliferation and induce cell death in AML cell lines, patient-derived leukemic blast and xenograft animal models, reaching best results in FLT3 mutants that carry other molecular pathways' alterations. Thus, its unique inhibition profile warrants EC-70124 as a promising agent for AML treatment based on its ability to interfere the complex oncogenic events activated in AML at several levels. Mol Cancer Ther; 17(3); 614–24. ©2018 AACR.
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Phase I Dose-Escalation Study of Anti-CTLA-4 Antibody Ipilimumab and Lenalidomide in Patients with Advanced Cancers
Preclinical data suggest that combining a checkpoint inhibition with immunomodulatory derivative can increase anticancer response. We designed a dose-escalation study using a 3 + 3 design to determine the safety, maximum tolerated dose (MTD) or recommended phase II dose (R2PD) and dose-limiting toxicities (DLT) of the anti–CTLA-4 antibody ipilimumab (1.5–3 mg/kg intravenously every 28 days x 4) and lenalidomide (10–25 mg orally daily for 21 of 28 days until disease progression or unacceptable toxicity) in advanced cancers. Total of 36 patients (Hodgkin lymphoma, 7; melanoma, 5; leiomyosarcoma, 4; renal cancer, 3; thyroid cancer, 3; other cancers, 14; median of 3 prior therapies) were enrolled. The MTD has not been reached and ipilimumab 3 mg/kg and lenalidomide 25 mg have been declared as R2PD. DLT were grade (G) 3 rash (3 patients) and G3 pancreatitis (1 patient). G3/4 drug-related toxicities other than DLT were G3 anemia (5 patients), G3 thromboembolism (2 patients), G3 thrombocytopenia, G3 rash, G3 hypopituitarism, G3 pneumonitis, G3 transaminitis, and G4 hypopituitarism (all in 1 patient). Eight patients had tumor shrinkage per immune-related response criteria (–79% to –2%) including a PR (–79% for 7.2+ months) in a refractory Hodgkin lymphoma. Using comprehensive genomic profiling, a total mutation burden (mutations/Mb) was evaluated in 17 patients, with one of the patients achieving a PR demonstrated intermediate mutation burden. In conclusion, combination of ipilimumab and lenalidomide is well tolerated and demonstrated preliminary signals of activity in patients with refractory Hodgkin lymphoma and other advanced cancers. Mol Cancer Ther; 17(3); 671–6. ©2017 AACR.
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Scavenger Receptor Type B1 and Lipoprotein Nanoparticle Inhibit Myeloid-Derived Suppressor Cells
Myeloid-derived suppressor cells (MDSC) are innate immune cells that potently inhibit T cells. In cancer, novel therapies aimed to activate T cells can be rendered ineffective due to the activity of MDSCs. Thus, targeted inhibition of MDSCs may greatly enhance T-cell–mediated antitumor immunity, but mechanisms remain obscure. Here we show, for the first time, that scavenger receptor type B-1 (SCARB1), a high-affinity receptor for spherical high-density lipoprotein (HDL), is expressed by MDSCs. Furthermore, we demonstrate that SCARB1 is specifically targeted by synthetic high-density lipoprotein-like nanoparticles (HDL NP), which reduce MDSC activity. Using in vitro T-cell proliferation assays, data show that HDL NPs specifically bind SCARB1 to inhibit MDSC activity. In murine cancer models, HDL NP treatment significantly reduces tumor growth, metastatic tumor burden, and increases survival due to enhanced adaptive immunity. Flow cytometry and IHC demonstrate that HDL NP–mediated suppression of MDSCs increased CD8+ T cells and reduced Treg cells in the metastatic tumor microenvironment. Using transgenic mice lacking SCARB1, in vivo data clearly show that the HDL NPs specifically target this receptor for suppressing MDSCs. Ultimately, our data provide a new mechanism and targeted therapy, HDL NPs, to modulate a critical innate immune cell checkpoint to enhance the immune response to cancer. Mol Cancer Ther; 17(3); 686–97. ©2017 AACR.
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Sex Differences in the Association of Diabetes With Cardiovascular Disease Outcomes Among African-American and White Participants in the Atherosclerosis Risk in Communities Study
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Breast Cancer Incidence and Exposure to Metalworking Fluid in a Cohort of Female Autoworkers
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Association of DNA Methylation-Based Biological Age With Health Risk Factors and Overall and Cause-Specific Mortality
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Possible Mediation by Methylation in Acute Inflammation Following Personal Exposure to Fine Particulate Air Pollution
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The Role of Mobile Genetic Elements in the Spread of Antimicrobial-Resistant Escherichia coli From Chickens to Humans in Small-Scale Production Poultry Operations in Rural Ecuador
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Understanding Causal Distributional and Subgroup Effects With the Instrumental Propensity Score
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Principled Approaches to Missing Data in Epidemiologic Studies
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Understanding Causal Distributional and Subgroup Effects With the Instrumental Propensity Score
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The Mental Health Benefits of Acquiring a Home in Older Age: A Fixed-Effects Analysis of Older US Adults
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When to Censor?
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Invited Commentary: “Bedroom Light Exposure at Night and the Incidence of Depressive Symptoms: A Longitudinal Study of the HEIJO-KYO Cohort”
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Therapy-Educated Mesenchymal Stem Cells Enrich for Tumor-Initiating Cells
Stromal cells residing in the tumor microenvironment contribute to the development of therapy resistance. Here we show that chemotherapy-educated mesenchymal stem cells (MSC) promote therapy resistance via cross-talk with tumor-initiating cells (TIC), a resistant tumor cell subset that initiates tumorigenesis and metastasis. In response to gemcitabine chemotherapy, MSCs colonized pancreatic adenocarcinomas in large numbers and resided in close proximity to TICs. Furthermore, gemcitabine-educated MSCs promoted the enrichment of TICs in vitro and enhance tumor growth in vivo. These effects were dependent on the secretion of CXCL10 by gemcitabine-educated MSCs and subsequent activation of the CXCL10–CXCR3 axis in TICs. In an orthotopic pancreatic tumor model, targeting TICs using nanovesicles (called nanoghosts) derived from MSC membranes and loaded with a CXCR3 antagonist enhanced therapy outcome and delayed tumor regrowth when administered in combination with gemcitabine. Overall, our results establish a mechanism through which MSCs promote chemoresistance, and propose a novel drug delivery system to target TICs and overcome this resistance.Significance: These results establish a mechanism by which mesenchyme stem cells in the tumor microenvironment promote chemoresistance, and they propose a novel drug delivery system to overcome this challenge. Cancer Res; 78(5); 1253–65. ©2018 AACR.
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Race Disparities in the Contribution of miRNA Isoforms and tRNA-Derived Fragments to Triple-Negative Breast Cancer
Triple-negative breast cancer (TNBC) is a breast cancer subtype characterized by marked differences between White and Black/African-American women. We performed a systems-level analysis on datasets from The Cancer Genome Atlas to elucidate how the expression patterns of mRNAs are shaped by regulatory noncoding RNAs (ncRNA). Specifically, we studied isomiRs, that is, isoforms of miRNAs, and tRNA-derived fragments (tRF). In normal breast tissue, we observed a marked cohesiveness in both the ncRNA and mRNA layers and the associations between them. This cohesiveness was widely disrupted in TNBC. Many mRNAs become either differentially expressed or differentially wired between normal breast and TNBC in tandem with isomiR or tRF dysregulation. The affected pathways included energy metabolism, cell signaling, and immune responses. Within TNBC, the wiring of the affected pathways with isomiRs and tRFs differed in each race. Multiple isomiRs and tRFs arising from specific miRNA loci (e.g., miR-200c, miR-21, the miR-17/92 cluster, the miR-183/96/182 cluster) and from specific tRNA loci (e.g., the nuclear tRNAGly and tRNALeu, the mitochondrial tRNAVal and tRNAPro) were strongly associated with the observed race disparities in TNBC. We highlight the race-specific aspects of transcriptome wiring by discussing in detail the metastasis-related MAPK and the Wnt/β-catenin signaling pathways, two of the many key pathways that were found differentially wired. In conclusion, by employing a data- and knowledge-driven approach, we comprehensively analyzed the normal and cancer transcriptomes to uncover novel key contributors to the race-based disparities of TNBC.Significance: This big data-driven study comparing normal and cancer transcriptomes uncovers RNA expression differences between Caucasian and African-American patients with triple-negative breast cancer that might help explain disparities in incidence and aggressive character. Cancer Res; 78(5); 1140–54. ©2017 AACR.
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FIH Is an Oxygen Sensor in Ovarian Cancer for G9a/GLP-Driven Epigenetic Regulation of Metastasis-Related Genes
The prolyl hydroxylase domain-containing proteins (PHD1-3) and the asparaginyl hydroxlyase factor inhibiting HIF (FIH) are oxygen sensors for hypoxia-inducible factor-driven transcription of hypoxia-induced genes, but whether these sensors affect oxygen-dependent epigenetic regulation more broadly is not known. Here, we show that FIH exerts an additional role as an oxygen sensor in epigenetic control by the histone lysine methyltransferases G9a and GLP. FIH hydroxylated and inhibited G9a and GLP under normoxia. When the FIH reaction was limited under hypoxia, G9a and GLP were activated and repressed metastasis suppressor genes, thereby triggering cancer cell migration and peritoneal dissemination of ovarian cancer xenografts. In clinical specimens of ovarian cancer, expression of FIH and G9a were reciprocally associated with patient outcomes. We also identified mutations of FIH target motifs in G9a and GLP, which exhibited excessive H3K9 methylation and facilitated cell invasion. This study provides insight into a new function of FIH as an upstream regulator of oxygen-dependent chromatin remodeling. It also implies that the FIH-G9a/GLP pathway could be a potential target for inhibiting hypoxia-induced cancer metastasis.Significance: These findings deepen understanding of oxygen-dependent gene regulation and cancer metastasis in response to hypoxia. Cancer Res; 78(5); 1184–99. ©2017 AACR.
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Loss of RASSF4 Expression in Multiple Myeloma Promotes RAS-Driven Malignant Progression
RAS mutations occur frequently in multiple myeloma (MM), but apart from driving progression, they can also stimulate antitumor effects by activating tumor-suppressive RASSF proteins. Although this family of death effector molecules are often silenced in cancers, functional data about RASSF proteins in MM are lacking. Here, we report that RASSF4 is downregulated during MM progression and correlates with a poor prognosis. Promoter methylation analysis in human cell lines revealed an inverse correlation between RASSF4 mRNA levels and methylation status. Epigenetic modulating agents restored RASSF4 expression. Enforced expression of RASSF4 induced G2-phase cell-cycle arrest and apoptosis in human cell lines, reduced primary MM cell viability, and blocked MM growth in vivo. Mechanistic investigations showed that RASSF4 linked RAS to several pro-death pathways, including those regulated by the kinases MST1, JNK, and p38. By activating MST1 and the JNK/c-Jun pathway, RASSF4 sensitized MM cells to bortezomib. Genetic or pharmacological elevation of RASSF4 levels increased the anti-MM effects of the clinical relevant MEK1/2 inhibitor trametinib. Kinome analysis revealed that this effect was mediated by concomitant activation of the JNK/c-Jun pathway along with inactivation of the MEK/ERK and PI3K/mTOR/Akt pathways. Overall, our findings establish RASSF4 as a tumor-suppressive hub in MM and provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib to treat patients with tumors exhibiting low RASSF4 expression.Significance: These findings provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib in patients with multiple myeloma whose tumors exhibit low RASSF4 expression. Cancer Res; 78(5); 1155–68. ©2017 AACR.
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O-GlcNAcylation of the Tumor Suppressor FOXO3 Triggers Aberrant Cancer Cell Growth
Posttranslational modifications of tumor suppressors can induce abnormal cell growth. Here, we identify site-specific O-GlcNAcylation as a critical block of FOXO3 that may abrogate a part of the p53 pathway, resulting in aberrant cancer cell growth. Of seven O-GlcNAcylation sites identified within the FOXO3 transactivation domain, we found that changes in O-GlcNAcylation at Ser284 modulated p21-mediated cancer cell growth. Overexpression of either O-GlcNAcylated FOXO3 (FOX-OV) or a Ser-to-Ala mutant (S284A) in PANC-1 cells indicated that S284 O-GlcNAc acts as a critical block of the FOXO tumor suppressor and induces proliferation in PANC-1 cancer cells by stimulating the MDM2-p53-p21 axis. Furthermore, S284A mutant cells lacking S284 O-GlcNAc and FOX-OV cells exhibited opposing MDM2-p53-p21 axis expression patterns at both the mRNA and protein levels. Thus, our study provides evidence to support a role for S284 O-GlcNAc as a critical block of FOXO3 to induce subsequent cancer cell growth via abrogation of the p53 regulatory circuit.Significance: These findings highlight a posttranslational mechanism for indirect abrogation of the p53 pathway, one that may occur with some frequency in human cancer cells. Cancer Res; 78(5); 1214–24. ©2018 AACR.
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Long Noncoding RNA pancEts-1 Promotes Neuroblastoma Progression through hnRNPK-Mediated {beta}-Catenin Stabilization
Long noncoding RNAs (lncRNA) play essential roles in tumor progression. However, the functions of lncRNAs in the tumorigenesis and aggressiveness of neuroblastoma still remain to be determined. Here, we report the identification of lncRNA pancEts-1 as a novel driver of neuroblastoma progression by using a public microarray dataset. LncRNA pancEts-1 promoted the growth, invasion, and metastasis of neuroblastoma cells in vitro and in vivo. Mechanistically, pancEts-1 bound to hnRNPK to facilitate its physical interaction with β-catenin, whereas hnRNPK stabilized the β-catenin by inhibiting proteasome-mediated degradation, resulting in transcriptional alteration of target genes associated with neuroblastoma progression. Both pancEts-1 and hnRNPK were upregulated in clinical neuroblastoma tissues, and were associated with unfavorable outcome of patients. Overall, our results define an oncogenic role of pancEts-1 in neuroblastoma progression through hnRNPK-mediated β-catenin stabilization, with potential implications for the clinical therapeutics of neuroblastoma.Significance: These findings reveal the oncogenic functions of a long noncoding RNA in neuroblastoma progression, offering a potential target for clinical therapeutics. Cancer Res; 78(5); 1169–83. ©2018 AACR.
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Nkx2.8 Inhibits Epithelial-Mesenchymal Transition in Bladder Urothelial Carcinoma via Transcriptional Repression of Twist1
Epithelial-to-mesenchymal transition (EMT) promotes metastasis, which is the main cause of bladder urothelial carcinoma–related death. Loss of the candidate tumor-suppressor gene Nkx2.8 has been associated with urothelial carcinoma lymph node metastasis. Here, we show that enforced expression of Nkx2.8 is sufficient to inhibit EMT, reduce motility, and blunt invasiveness of urothelial carcinoma cells. Mechanistic investigations showed that Nkx2.8 negatively regulated expression of the EMT inducer Twist1 in urothelial carcinoma cells, at both the level of mRNA and protein accumulation. Nkx2.8 bound directly to the promoter region of this gene and transcriptionally repressed its expression. Twist1 upregulation reversed EMT inhibition by Nkx2.8, restoring the invasive phenotype of urothelial carcinoma cells. In clinical urothelial carcinoma specimens, expression of Nkx2.8 inversely correlated with Twist1 expression, and urothelial carcinoma patients with Nkx2.8 positivity and low Twist1 expression displayed the best prognosis. Our findings highlight the Nkx2.8–Twist1 axis as candidate target for therapeutic intervention in advanced urothelial carcinoma.Significance: These findings highlight a novel EMT signaling axis as a candidate target for therapeutic intervention in advanced urothelial carcinomas. Cancer Res; 78(5); 1241–52. ©2018 AACR.
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Deletion of the von Hippel-Lindau Gene in Hemangioblasts Causes Hemangioblastoma-like Lesions in Murine Retina
von Hippel-Lindau (VHL) disease is an autosomal-dominant tumor predisposition syndrome characterized by the development of highly vascularized tumors and cysts. LOH of the VHL gene results in aberrant upregulation of hypoxia-inducible factors (HIF) and has been associated with tumor formation. Hemangioblastomas of the central nervous system and retina represent the most prevalent VHL-associated tumors, but no VHL animal model has reproduced retinal capillary hemangioblastomas (RCH), the hallmark lesion of ocular VHL. Here we report our work in developing a murine model of VHL-associated RCH by conditionally inactivating Vhl in a hemangioblast population using a Scl-Cre-ERT2 transgenic mouse line. In transgenic mice carrying the conditional allele and the Scl-Cre-ERT2 allele, 64% exhibited various retinal vascular anomalies following tamoxifen induction. Affected Vhl-mutant mice demonstrated retinal vascular lesions associated with prominent vasculature, anomalous capillary networks, hemorrhage, exudates, and localized fibrosis. Histologic analyses showed RCH-like lesions characterized by tortuous, dilated vasculature surrounded by "tumorlet" cell cluster and isolated foamy stromal cells, which are typically associated with RCH. Fluorescein angiography suggested increased vascular permeability of the irregular retinal vasculature and hemangioblastoma-like lesions. Vhl deletion was detected in "tumorlet" cells via microdissection. Our findings provide a phenotypic recapitulation of VHL-associated RCH in a murine model that may be useful to study RCH pathogenesis and therapeutics aimed at treating ocular VHL.Significance: This study describes a model that phenotypically recapitulates a form of retinal pathogenesis that is driven by genetic loss of the VHL tumor suppressor, providing a useful tool for its study and therapeutic intervention. Cancer Res; 78(5); 1266–74. ©2018 AACR.
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Combined Mutation of Apc, Kras, and Tgfbr2 Effectively Drives Metastasis of Intestinal Cancer
Colorectal cancer is driven by the accumulation of driver mutations, but the contributions of specific mutations to different steps in malignant progression are not fully understood. In this study, we generated mouse models harboring different combinations of key colorectal cancer driver mutations (Apc, Kras, Tgfbr2, Trp53, Fbxw7) in intestinal epithelial cells to comprehensively investigate their roles in the development of primary tumors and metastases. ApcΔ716 mutation caused intestinal adenomas and combination with Trp53R270H mutation or Tgfbr2 deletion induced submucosal invasion. The addition of KrasG12D mutation yielded epithelial–mesenchymal transition (EMT)-like morphology and lymph vessel intravasation of the invasive tumors. In contrast, combinations of ApcΔ716 with KrasG12D and Fbxw7 mutation were insufficient for submucosal invasion, but still induced EMT-like histology. Studies using tumor-derived organoids showed that KrasG12D was critical for liver metastasis following splenic transplantation, when this mutation was combined with either ApcΔ716 plus Trp53R270H or Tgfbr2 deletion, with the highest incidence of metastasis displayed by tumors with a ApcΔ716 KrasG12D Tgfbr2−/− genotype. RNA sequencing analysis of tumor organoids defined distinct gene expression profiles characteristic for the respective combinations of driver mutations, with upregulated genes in ApcΔ716 KrasG12D Tgfbr2−/− tumors found to be similarly upregulated in specimens of human metastatic colorectal cancer. Our results show how activation of Wnt and Kras with suppression of TGFβ signaling in intestinal epithelial cells is sufficient for colorectal cancer metastasis, with possible implications for the development of metastasis prevention strategies.Significance: These findings illuminate how key driver mutations in colon cancer cooperate to drive the development of metastatic disease, with potential implications for the development of suitable prevention strategies. Cancer Res; 78(5); 1334–46. ©2017 AACR.
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Inactivation of Cancer-Associated-Fibroblasts Disrupts Oncogenic Signaling in Pancreatic Cancer Cells and Promotes Its Regression
Resident fibroblasts that contact tumor epithelial cells (TEC) can become irreversibly activated as cancer-associated-fibroblasts (CAF) that stimulate oncogenic signaling in TEC. In this study, we evaluated the cross-talk between CAF and TEC isolated from tumors generated in a mouse model of KRAS/mut p53-induced pancreatic cancer (KPC mice). Transcriptomic profiling conducted after treatment with the anticancer compound Minnelide revealed deregulation of the TGFβ signaling pathway in CAF, resulting in an apparent reversal of their activated state to a quiescent, nonproliferative state. TEC exposed to media conditioned by drug-treated CAFs exhibited a decrease in oncogenic signaling, as manifested by downregulation of the transcription factor Sp1. This inhibition was rescued by treating TEC with TGFβ. Given promising early clinical studies with Minnelide, our findings suggest that approaches to inactivate CAF and prevent tumor–stroma cross-talk may offer a viable strategy to treat pancreatic cancer.Significance: In an established mouse model of pancreatic cancer, administration of the promising experimental drug Minnelide was found to actively deplete reactive stromal fibroblasts and to trigger tumor regression, with implications for stromal-based strategies to attack this disease. Cancer Res; 78(5); 1321–33. ©2018 AACR.
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Brain-Mimetic 3D Culture Platforms Allow Investigation of Cooperative Effects of Extracellular Matrix Features on Therapeutic Resistance in Glioblastoma
Glioblastoma (GBM) tumors exhibit potentially actionable genetic alterations against which targeted therapies have been effective in treatment of other cancers. However, these therapies have largely failed in GBM patients. A notable example is kinase inhibitors of EGFR, which display poor clinical efficacy despite overexpression and/or mutation of EGFR in >50% of GBM. In addressing this issue, preclinical models may be limited by the inability to accurately replicate pathophysiologic interactions of GBM cells with unique aspects of the brain extracellular matrix (ECM), which is relatively enriched in hyaluronic acid (HA) and flexible. In this study, we present a brain-mimetic biomaterial ECM platform for 3D culturing of patient-derived GBM cells, with improved pathophysiologic properties as an experimental model. Compared with orthotopic xenograft assays, the novel biomaterial cultures we developed better preserved the physiology and kinetics of acquired resistance to the EGFR inhibition than gliomasphere cultures. Orthogonal modulation of both HA content and mechanical properties of biomaterial scaffolds was required to achieve this result. Overall, our findings show how specific interactions between GBM cell receptors and scaffold components contribute significantly to resistance to the cytotoxic effects of EGFR inhibition.Significance: Three-dimensional culture scaffolds of glioblastoma provide a better physiological representation over current methods of patient-derived cell culture and xenograft models. Cancer Res; 78(5); 1358–70. ©2017 AACR.
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Immunotherapy of Myelodysplastic Syndrome: You Can Run, but You Can't Hide
The hypomethylating agent decitabine induces expression of the cancer/testis antigen NY-ESO-1 in the myeloid cells of patients with myelodysplastic syndrome (MDS). Patients with MDS treated with decitabine and an NY-ESO-1 vaccine developed NY-ESO-1–specific T-cell responses directed against their abnormal myeloid cells, raising hopes for combinatorial immunotherapy of this disease. Clin Cancer Res; 24(5); 991–3. ©2017 AACR.
See related article by Griffiths et al., p. 1019
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HSP27-Mediated Extracellular and Intracellular Signaling Pathways Synergistically Confer Chemoresistance in Squamous Cell Carcinoma of Tongue
Purpose: Squamous cell carcinoma of tongue (SCCT) is the most common type of oral cavity carcinoma. Chemoresistance in SCCT is common, and the underlying mechanism remains largely unknown. We aimed to identify key molecules and signaling pathways mediating chemoresistance in SCCT.
Experimental Design: Using a proteomic approach, we found that the HSP27 was a potential mediator for chemoresistance in SCCT cells. To further validate this role of HSP27, we performed various mechanistic studies using in vitro and in vivo models as well as serum and tissue samples from SCCT patients.
Results: The HSP27 protein level was significantly increased in the multidrug-resistant SCCT cells and cell culture medium. Both HSP27 knockdown and anti-HSP27 antibody treatment reversed chemoresistance. Inversely, both HSP27 overexpression and recombinant human HSP27 protein treatment enhanced chemoresistance. Moreover, chemotherapy significantly induced HSP27 protein expression in both SCCT cells and their culture medium, as well as in tumor tissues and serum of SCCT patients. HSP27 overexpression predicts a poor outcome for SCCT patients receiving chemotherapy. Mechanically, extracellular HSP27 binds to TLR5 and then activates NF-B signaling to maintain SCCT cell survival. TLR5 knockdown or restored IBα protein level disrupts extracellular HSP27-induced NF-B transactivation and chemoresistance. Moreover, intracellular HSP27 binds to BAX and BIM to repress their translocation to mitochondrion and subsequent cytochrome C release upon chemotherapy, resulting in inhibition of the mitochondrial apoptotic pathway.
Conclusions: HSP27 plays a pivotal role in chemoresistance of SCCT cells via a synergistic extracellular and intracellular signaling. HSP27 may represent a potential biomarker and therapeutic target for precision SCCT treatment. Clin Cancer Res; 24(5); 1163–75. ©2017 AACR.
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Biomarker-Stratified Phase III Clinical Trials: Enhancement with a Subgroup-Focused Sequential Design
Among various design approaches to phase III clinical trials with a predictive biomarker, the marker-stratified all-comers design is advantageous because it allows for establishing the utility of both treatment and biomarker, but it is often criticized for requiring large sample sizes, as the design includes both marker-positive and marker-negative patients. In this article, we propose a simple but flexible subgroup-focused design for marker-stratified trials that allow both sequential assessment across marker-defined subgroups and adaptive subgroup selection while retaining an assessment using the entire patient cohort at the final analysis stage, possibly using established marker-based multiple testing procedures. Numerical evaluations indicate that the proposed marker-stratified design has a robustness property in preserving statistical power for detecting various profiles of treatment effects across the subgroups while effectively reducing the number of randomized patients in the marker-negative subgroup with presumably limited treatment efficacy. In contrast, the traditional all-comers and sequential enrichment designs could suffer from low statistical power for some possible profiles of treatment effects. The latter also needs long study durations and a large number of marker-screened patients. We also provide an application to SWOG S0819, a trial to assess the role of cetuximab in treating non–small cell lung cancers. These evaluations indicate that the proposed subgroup-focused approach can enhance the efficiency of the marker-stratified design for definitive evaluation of treatment and biomarker in phase III clinical trials. Clin Cancer Res; 24(5); 994–1001. ©2017 AACR.
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Acetylsalicylic Acid Governs the Effect of Sorafenib in RAS-Mutant Cancers
Purpose: Identify and characterize novel combinations of sorafenib with anti-inflammatory painkillers to target difficult-to-treat RAS-mutant cancer.
Experimental Design: The cytotoxicity of acetylsalicylic acid (aspirin) in combination with the multikinase inhibitor sorafenib (Nexavar) was assessed in RAS-mutant cell lines in vitro. The underlying mechanism for the increased cytotoxicity was investigated using selective inhibitors and shRNA-mediated gene knockdown. In vitro results were confirmed in RAS-mutant xenograft mouse models in vivo.
Results: The addition of aspirin but not isobutylphenylpropanoic acid (ibruprofen) or celecoxib (Celebrex) significantly increased the in vitro cytotoxicity of sorafenib. Mechanistically, combined exposure resulted in increased BRAF/CRAF dimerization and the simultaneous hyperactivation of the AMPK and ERK pathways. Combining sorafenib with other AMPK activators, such as metformin or A769662, was not sufficient to decrease cell viability due to sole activation of the AMPK pathway. The cytotoxicity of sorafenib and aspirin was blocked by inhibition of the AMPK or ERK pathways through shRNA or via pharmacologic inhibitors of RAF (LY3009120), MEK (trametinib), or AMPK (compound C). The combination was found to be specific for RAS/RAF–mutant cells and had no significant effect in RAS/RAF–wild-type keratinocytes or melanoma cells. In vivo treatment of human xenografts in NSG mice with sorafenib and aspirin significantly reduced tumor volume compared with each single-agent treatment.
Conclusions: Combination sorafenib and aspirin exerts cytotoxicity against RAS/RAF–mutant cells by simultaneously affecting two independent pathways and represents a promising novel strategy for the treatment of RAS-mutant cancers. Clin Cancer Res; 24(5); 1090–102. ©2017 AACR.
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Immunotherapy of Lymphoma and Myeloma: Facts and Hopes
Immune checkpoint blockade has driven a revolution in modern oncology, and robust drug development of immune checkpoint inhibitors is underway in both solid tumors and hematologic malignancies. High response rates to programmed cell death 1 (PD-1) blockade using nivolumab or pembrolizumab in classical Hodgkin lymphoma (cHL) and several variants of non-Hodgkin lymphoma (NHL) revealed an intrinsic biological sensitivity to this approach, and work is ongoing exploring combinations with immune checkpoint inhibitors in both cHL and NHL. There are also preliminary data suggesting antitumor efficacy of PD-1 inhibitors used in combination with immunomodulatory drugs in multiple myeloma, and effects of novel monoclonal antibody therapies on the tumor microenvironment may lead to synergy with checkpoint blockade. Although immune checkpoint inhibitors are generally well tolerated, clinicians must use caution and remain vigilant when treating patients with these agents in order to identify immune-related toxicities and prevent treatment-related morbidity and mortality. Autologous stem cell transplant is a useful tool for treatment of hematologic malignancies and has potential as a platform for use of immune checkpoint inhibitors. An important safety signal has emerged surrounding the risk of graft-versus-host disease associated with use of PD-1 inhibitors before and after allogeneic stem cell transplant. We aim to discuss the facts known to date in the use of immune checkpoint inhibitors for patients with lymphoid malignancies and our hopes for expanding the benefits of immunotherapy to patients in the future. Clin Cancer Res; 24(5); 1002–10. ©2017 AACR.
http://ift.tt/2EZS8fa
Activation of 4-1BB on Liver Myeloid Cells Triggers Hepatitis via an Interleukin-27-Dependent Pathway
Purpose: Agonist antibodies targeting the T-cell costimulatory receptor 4-1BB (CD137) are among the most effective immunotherapeutic agents across preclinical cancer models. In the clinic, however, development of these agents has been hampered by dose-limiting liver toxicity. Lack of knowledge of the mechanisms underlying this toxicity has limited the potential to separate 4-1BB agonist–driven tumor immunity from hepatotoxicity.
Experimental Design: The capacity of 4-1BB agonist antibodies to induce liver toxicity was investigated in immunocompetent mice, with or without coadministration of checkpoint blockade, via (i) measurement of serum transaminase levels, (ii) imaging of liver immune infiltrates, and (iii) qualitative and quantitative assessment of liver myeloid and T cells via flow cytometry. Knockout mice were used to clarify the contribution of specific cell subsets, cytokines, and chemokines.
Results: We find that activation of 4-1BB on liver myeloid cells is essential to initiate hepatitis. Once activated, these cells produce interleukin-27 that is required for liver toxicity. CD8 T cells infiltrate the liver in response to this myeloid activation and mediate tissue damage, triggering transaminase elevation. FoxP3+ regulatory T cells limit liver damage, and their removal dramatically exacerbates 4-1BB agonist–induced hepatitis. Coadministration of CTLA-4 blockade ameliorates transaminase elevation, whereas PD-1 blockade exacerbates it. Loss of the chemokine receptor CCR2 blocks 4-1BB agonist hepatitis without diminishing tumor-specific immunity against B16 melanoma.
Conclusions: 4-1BB agonist antibodies trigger hepatitis via activation and expansion of interleukin-27–producing liver Kupffer cells and monocytes. Coadministration of CTLA-4 and/or CCR2 blockade may minimize hepatitis, but yield equal or greater antitumor immunity. Clin Cancer Res; 24(5); 1138–51. ©2018 AACR.
http://ift.tt/2CPXio4
Ipilimumab plus Lenalidomide after Allogeneic and Autologous Stem Cell Transplantation for Patients with Lymphoid Malignancies
Purpose: Prevention or treatment of relapsed lymphoid malignancies after hematopoietic stem cell transplantation (HSCT) requires novel strategies. We hypothesized that antitumor–cell responses could be enhanced by the addition of lenalidomide to the cytotoxic T-lymphocyte–associated protein 4 inhibitor ipilimumab.
Experimental Design: We conducted a phase II investigator-initiated trial to assess the safety and activity of ipilimumab and lenalidomide in patients with lymphoid malignancies that relapsed after allogeneic HSCT and in high-risk patients after autologous HSCT. Patients received 10 mg of oral lenalidomide daily for 21 days followed by intravenous ipilimumab at 3 mg/kg bodyweight. The regimen was repeated 4 weeks later for a total of four treatments.
Results: We enrolled 17 patients (10 allogeneic and seven autologous transplant recipients). Immune-mediated toxicity was limited to one patient with asymptomatic hypothyroidism and one with dermatitis in the allogeneic and autologous groups, respectively. One allogeneic transplant recipient had a flare of prior GVHD while taking lenalidomide that precluded further treatment. All others finished treatment without GVHD. Four of 10 patients in the allogeneic group had complete responses (three of which were durable at 19+, 21+, and 32+ months), and three had partial responses. The disease in six of seven patients in the autologous group remains in remission. The groups had similar immune responses, including a two- to threefold increase in inducible ICOS+CD4+FoxP3– T-cell number.
Conclusions: Our early-phase data suggested that ipilimumab plus lenalidomide is well tolerated after HSCT. Adverse events did not differ significantly between the allogeneic and autologous groups. Clin Cancer Res; 24(5); 1011–8. ©2017 AACR.
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Alteration of the Tumor Stroma Using a Consensus DNA Vaccine Targeting Fibroblast Activation Protein (FAP) Synergizes with Antitumor Vaccine Therapy in Mice
Purpose: Fibroblast activation protein (FAP) is overexpressed in cancer-associated fibroblasts and is an interesting target for cancer immune therapy, with prior studies indicating a potential to affect the tumor stroma. Our aim was to extend this earlier work through the development of a novel FAP immunogen with improved capacity to break tolerance for use in combination with tumor antigen vaccines.
Experimental Design: We used a synthetic consensus (SynCon) sequence approach to provide MHC class II help to support breaking of tolerance. We evaluated immune responses and antitumor activity of this novel FAP vaccine in preclinical studies, and correlated these findings to patient data.
Results: This SynCon FAP DNA vaccine was capable of breaking tolerance and inducing both CD8+ and CD4+ immune responses. In genetically diverse, outbred mice, the SynCon FAP DNA vaccine was superior at breaking tolerance compared with a native mouse FAP immunogen. In several tumor models, the SynCon FAP DNA vaccine synergized with other tumor antigen–specific DNA vaccines to enhance antitumor immunity. Evaluation of the tumor microenvironment showed increased CD8+ T-cell infiltration and a decreased macrophage infiltration driven by FAP immunization. We extended this to patient data from The Cancer Genome Atlas, where we find high FAP expression correlates with high macrophage and low CD8+ T-cell infiltration.
Conclusions: These results suggest that immune therapy targeting tumor antigens in combination with a microconsensus FAP vaccine provides two-fisted punch-inducing responses that target both the tumor microenvironment and tumor cells directly. Clin Cancer Res; 24(5); 1190–201. ©2018 AACR.
http://ift.tt/2COVig4
NY-ESO-1 Vaccination in Combination with Decitabine Induces Antigen-Specific T-lymphocyte Responses in Patients with Myelodysplastic Syndrome
Purpose: Treatment options are limited for patients with high-risk myelodysplastic syndrome (MDS). The azanucleosides, azacitidine and decitabine, are first-line therapy for MDS that induce promoter demethylation and gene expression of the highly immunogenic tumor antigen NY-ESO-1. We demonstrated that patients with acute myeloid leukemia (AML) receiving decitabine exhibit induction of NY-ESO-1 expression in circulating blasts. We hypothesized that vaccinating against NY-ESO-1 in patients with MDS receiving decitabine would capitalize upon induced NY-ESO-1 expression in malignant myeloid cells to provoke an NY-ESO-1–specific MDS-directed cytotoxic T-cell immune response.
Experimental Design: In a phase I study, 9 patients with MDS received an HLA-unrestricted NY-ESO-1 vaccine (CDX-1401 + poly-ICLC) in a nonoverlapping schedule every four weeks with standard-dose decitabine.
Results: Analysis of samples serially obtained from the 7 patients who reached the end of the study demonstrated induction of NY-ESO-1 expression in 7 of 7 patients and NY-ESO-1–specific CD4+ and CD8+ T-lymphocyte responses in 6 of 7 and 4 of 7 of the vaccinated patients, respectively. Myeloid cells expressing NY-ESO-1, isolated from a patient at different time points during decitabine therapy, were capable of activating a cytotoxic response from autologous NY-ESO-1–specific T lymphocytes. Vaccine responses were associated with a detectable population of CD141Hi conventional dendritic cells, which are critical for the uptake of NY-ESO-1 vaccine and have a recognized role in antitumor immune responses.
Conclusions: These data indicate that vaccination against induced NY-ESO-1 expression can produce an antigen-specific immune response in a relatively nonimmunogenic myeloid cancer and highlight the potential for induced antigen-directed immunotherapy in a group of patients with limited options. Clin Cancer Res; 24(5); 1019–29. ©2017 AACR.
See related commentary by Fuchs, p. 991
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Synergistic Targeting of the Regulatory and Catalytic Subunits of PI3K{delta} in Mature B-cell Malignancies
Purpose: Aberrant activation of the B-cell receptor (BCR) is implicated in the pathogenesis of mature B-cell tumors, a concept validated in part by the clinical success of inhibitors of the BCR-related kinases BTK (Bruton's tyrosine kinase) and PI3K. These inhibitors have limitations, including the paucity of complete responses, acquired resistance, and toxicity. Here, we examined the mechanism by which the cyclic-AMP/PDE4 signaling axis suppresses PI3K, toward identifying a novel mechanism-based combinatorial strategy to attack BCR-dependency in mature B-cell malignancies.
Experimental Design: We used in vitro and in vivo diffuse large B-cell lymphoma (DLBCL) cell lines and primary chronic lymphocytic leukemia (CLL) samples to preclinically evaluate the effects of the combination of the FDA-approved phosphodiesterase 4 (PDE4) inhibitor roflumilast and idelalisib on cell survival and tumor growth. Genetic models of gain- and loss-of-function were used to map multiple signaling intermediaries downstream of the BCR.
Results: Roflumilast elevates the intracellular levels of cyclic-AMP and synergizes with idelalisib in suppressing tumor growth and PI3K activity. Mechanistically, we show that roflumilast suppresses PI3K by inhibiting BCR-mediated activation of the P85 regulatory subunit, distinguishing itself from idelalisib, an ATP-competitive inhibitor of the catalytic P110 subunit. Using genetic models, we linked the PDE4-regulated modulation of P85 activation to the oncogenic kinase SYK.
Conclusions: These data demonstrate that roflumilast and idelalisib suppress PI3K by distinct mechanisms, explaining the basis for their synergism, and suggest that the repurposing of PDE4 inhibitors to treat BCR-dependent malignancies is warranted. Clin Cancer Res; 24(5); 1103–13. ©2017 AACR.
http://ift.tt/2CPXaoA
The Impact of Smoking and TP53 Mutations in Lung Adenocarcinoma Patients with Targetable Mutations--The Lung Cancer Mutation Consortium (LCMC2)
Purpose: Multiplex genomic profiling is standard of care for patients with advanced lung adenocarcinomas. The Lung Cancer Mutation Consortium (LCMC) is a multi-institutional effort to identify and treat oncogenic driver events in patients with lung adenocarcinomas.
Experimental Design: Sixteen U.S. institutions enrolled 1,367 patients with lung cancer in LCMC2; 904 were deemed eligible and had at least one of 14 cancer-related genes profiled using validated methods including genotyping, massively parallel sequencing, and IHC.
Results: The use of targeted therapies in patients with EGFR, ERBB2, or BRAF p.V600E mutations, ALK, ROS1, or RET rearrangements, or MET amplification was associated with a survival increment of 1.5 years compared with those with such mutations not receiving targeted therapy, and 1.0 year compared with those lacking a targetable driver. Importantly, 60 patients with a history of smoking derived similar survival benefit from targeted therapy for alterations in EGFR/ALK/ROS1, when compared with 75 never smokers with the same alterations. In addition, coexisting TP53 mutations were associated with shorter survival among patients with EGFR, ALK, or ROS1 alterations.
Conclusion: Patients with adenocarcinoma of the lung and an oncogenic driver mutation treated with effective targeted therapy have a longer survival, regardless of prior smoking history. Molecular testing should be performed on all individuals with lung adenocarcinomas irrespective of clinical characteristics. Routine use of massively parallel sequencing enables detection of both targetable driver alterations and tumor suppressor gene and other alterations that have potential significance for therapy selection and as predictive markers for the efficacy of treatment. Clin Cancer Res; 24(5); 1038–47. ©2017 AACR.
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