Publications by authors named "Thomas M Roberts"

104 Publications

RNAi-Based Approaches for Pancreatic Cancer Therapy.

Pharmaceutics 2021 Oct 8;13(10). Epub 2021 Oct 8.

Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea.

Pancreatic cancer is one of the most lethal forms of cancer, predicted to be the second leading cause of cancer-associated death by 2025. Despite intensive research for effective treatment strategies and novel anticancer drugs over the past decade, the overall patient survival rate remains low. RNA interference (RNAi) is capable of interfering with expression of specific genes and has emerged as a promising approach for pancreatic cancer because genetic aberrations and dysregulated signaling are the drivers for tumor formation and the stromal barrier to conventional therapy. Despite its therapeutic potential, RNA-based drugs have remaining hurdles such as poor tumor delivery and susceptibility to serum degradation, which could be overcome with the incorporation of nanocarriers for clinical applications. Here we summarize the use of small interfering RNA (siRNA) and microRNA (miRNA) in pancreatic cancer therapy in preclinical reports with approaches for targeting either the tumor or tumor microenvironment (TME) using various types of nanocarriers. In these studies, inhibition of oncogene expression and induction of a tumor suppressive response in cancer cells and surrounding immune cells in TME exhibited a strong anticancer effect in pancreatic cancer models. The review discusses the remaining challenges and prospective strategies suggesting the potential of RNAi-based therapeutics for pancreatic cancer.
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http://dx.doi.org/10.3390/pharmaceutics13101638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8541396PMC
October 2021

The Mediator captures CDK7, an attractive transcriptional target in cancer.

Cancer Cell 2021 Sep 19;39(9):1184-1186. Epub 2021 Aug 19.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Departments of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA. Electronic address:

Cyclin-dependent kinase 7 (CDK7) is implicated in regulating the expression of cancer-dependent genes, and multiple CDK7-targeted therapies are currently under clinical investigation. Three recent studies elucidate the structure of human transcription machinery, offering vital mechanistic insights into CDK7 function and a potential pharmacodynamic marker of CDK7 activity in tumors.
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http://dx.doi.org/10.1016/j.ccell.2021.07.021DOI Listing
September 2021

Statin-mediated inhibition of RAS prenylation activates ER stress to enhance the immunogenicity of KRAS mutant cancer.

J Immunother Cancer 2021 07;9(7)

Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea

Background: Statins preferentially promote tumor-specific apoptosis by depleting isoprenoid such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate. However, statins have not yet been approved for clinical cancer treatment due, in part, to poor understanding of molecular determinants on statin sensitivity. Here, we investigated the potential of statins to elicit enhanced immunogenicity of -mutant ( ) tumors.

Methods: The immunogenicity of treated cancer cells was determined by western blot, flow cytometry and confocal microscopy. The immunotherapeutic efficacy of mono or combination therapy using statin was assessed in tumor models, including syngeneic colorectal cancer and genetically engineered lung and pancreatic tumors. Using NanoString analysis, we analyzed how statin influenced the gene signatures associated with the antigen presentation of dendritic cells in vivo and evaluated whether statin could induce CD8+ T-cell immunity. Multiplex immunohistochemistry was performed to better understand the complicated tumor-immune microenvironment.

Results: Statin-mediated inhibition of prenylation provoked severe endoplasmic reticulum (ER) stress by attenuating the anti-ER stress effect of mutation, thereby resulting in the immunogenic cell death (ICD) of cancer cells. Moreover, statin-mediated ICD enhanced the cross-priming ability of dendritic cells, thereby provoking CD8+ T-cell immune responses against tumors. Combination therapy using statin and oxaliplatin, an ICD inducer, significantly enhanced the immunogenicity of tumors and promoted tumor-specific immunity in syngeneic and genetically engineered tumor models. Along with immune-checkpoint inhibitors, the abovementioned combination therapy overcame resistance to PD-1 blockade therapies, improving the survival rate of tumor models.

Conclusions: Our findings suggest that mutation could be a molecular target for statins to elicit potent tumor-specific immunity.
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http://dx.doi.org/10.1136/jitc-2021-002474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8327837PMC
July 2021

TMTpro-18plex: The Expanded and Complete Set of TMTpro Reagents for Sample Multiplexing.

J Proteome Res 2021 05 26;20(5):2964-2972. Epub 2021 Apr 26.

Department of Cell Biology, Harvard Medical School, Boston 02115, Massachusetts, United States.

The development of the TMTpro-16plex series expanded the breadth of commercial isobaric tagging reagents by nearly 50% over classic TMT-11plex. In addition to the described 16plex reagents, the proline-based TMTpro molecule can accommodate two additional combinations of heavy carbon and nitrogen isotopes. Here, we introduce the final two labeling reagents, TMTpro-134C and TMTpro-135N, which permit the simultaneous global protein profiling of 18 samples with essentially no missing values. For example, six conditions with three biological replicates can now be perfectly accommodated. We showcase the 18plex reagent set by profiling the proteome and phosphoproteome of a pair of isogenic mammary epithelial cell lines under three conditions in triplicate. We compare the depth and quantitative performance of this data set with a TMTpro-16plex experiment in which two samples were omitted. Our analysis revealed similar numbers of quantified peptides and proteins, with high quantitative correlation. We interrogated further the TMTpro-18plex data set by highlighting changes in protein abundance profiles under different conditions in the isogenic cell lines. We conclude that TMTpro-18plex further expands the sample multiplexing landscape, allowing for complex and innovative experimental designs.
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http://dx.doi.org/10.1021/acs.jproteome.1c00168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210943PMC
May 2021

The role of the PIK3CA gene in the development and aging of the brain.

Sci Rep 2021 01 11;11(1):291. Epub 2021 Jan 11.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA.

The CLOVES syndrome is an overgrowth disease arising from mosaic activating somatic mutations in the PIK3CA gene. These mutations occur during fetal development producing malformation and overgrowth of a variety of tissues. It has recently been shown that treatment with low doses of a selective inhibitor of Class I PI3K catalytic subunit p110α, the protein product of the PIK3CA gene, can yield dramatic therapeutic benefits for patients with CLOVES and PROS (a spectrum of PIK3CA-related overgrowth syndromes). To assess the long-term effects of moderate loses of p110α activity, we followed development and growth of mice with heterozygous loss of p110α (Pik3ca) over their entire lifetimes, paying particular attention to effects on the brain. While homozygous deletion of the Pik3ca gene is known to result in early embryonic lethality, these Pik3ca mice displayed a longer lifespan compared to their wild-type littermates. These mice appeared normal, exhibited no obvious behavioral abnormalities, and no body weight changes. However, their brains showed a significant reduction in size and weight. Notably, mice featuring deletion of one allele of Pik3ca only in the brain also showed gradually reduced brain size and weight. Mechanistically, either deletion of p110α or pharmacological inhibition of p110α activity reduced neurosphere size, but not numbers, in vitro, suggesting that p110α activity is critical for neuronal stem cells. The phenotypes observed in our two genetically engineered mouse models suggest that the sustained pharmacological inhibition of the PIK3CA activity in human patients might have both beneficial and harmful effects, and future treatments may need to be deployed in a way to avoid or minimize adverse effects.
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http://dx.doi.org/10.1038/s41598-020-79416-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801510PMC
January 2021

Divergent Roles of PI3K Isoforms in PTEN-Deficient Glioblastomas.

Cell Rep 2020 09;32(13):108196

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02215, USA; Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. Electronic address:

Loss of PTEN, the negative regulator of PI3K activity, is frequent in glioblastomas (GBMs). However, the role of the two major PI3K isoforms, p110α and p110β, in PTEN-deficient gliomagenesis remains unknown. We show that PTEN-deficient GBM largely depends on p110α for proliferation and p110β for migration. Genetic ablation of either isoform delays tumor progression in mice, but only ablating both isoforms completely blocks GBM driven by the concurrent ablation of Pten and p53. BKM120 (buparlisib) treatment only modestly prolongs survival in mice bearing intracranial Pten/p53 null tumors due to partial pathway inhibition. BKM120 extends the survival of mice bearing intracranial tumors in which p110β, but not p110α, has been genetically ablated in the Pten/p53 null glioma, indicating that BKM120 fails to inhibit p110β effectively. Our study suggests that the failure of PI3K inhibitors in GBM may be due to insufficient inhibition of p110β and indicates a need to develop brain-penetrant p110α/β inhibitors.
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http://dx.doi.org/10.1016/j.celrep.2020.108196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7571617PMC
September 2020

PIK3CA C-terminal frameshift mutations are novel oncogenic events that sensitize tumors to PI3K-α inhibition.

Proc Natl Acad Sci U S A 2020 09 14;117(39):24427-24433. Epub 2020 Sep 14.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115;

hotspot mutation is well established as an oncogenic driver event in cancer and its durable and efficacious inhibition is a focus in the development and testing of clinical cancer therapeutics. However, hundreds of cancer-associated mutations remain uncharacterized, their sensitivity to PI3K inhibitors unknown. Here, we describe a series of C-terminal mutations, primarily nucleotide insertions, that produce a frame-shifted protein product with an extended C terminus. We report that these mutations occur at a low frequency across multiple cancer subtypes, including breast, and are sufficient to drive oncogenic transformation in vitro and in vivo. We demonstrate that the oncogenicity of these mutant p110α proteins is dependent on p85 but not Ras association. P110α-selective pharmacologic inhibition blocks transformation in cells and mammary tumors characterized by C-terminal mutation. Taken together, these results suggest patients with breast and other tumors characterized by C-terminal frameshift mutations may derive benefit from p110α-selective inhibitors, including the recently FDA-approved alpelisib.
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http://dx.doi.org/10.1073/pnas.2000060117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533832PMC
September 2020

Multiplex Immunofluorescence in Formalin-Fixed Paraffin-Embedded Tumor Tissue to Identify Single-Cell-Level PI3K Pathway Activation.

Clin Cancer Res 2020 11 10;26(22):5903-5913. Epub 2020 Sep 10.

Department of Oncologic Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts.

Purpose: Identifying cancers with high PI3K pathway activity is critical for treatment selection and eligibility into clinical trials of PI3K inhibitors. Assessments of tumor signaling pathway activity need to consider intratumoral heterogeneity and multiple regulatory nodes.

Experimental Design: We established a novel, mechanistically informed approach to assessing tumor signaling pathways by quantifying single-cell-level multiplex immunofluorescence using custom algorithms. In a proof-of-concept study, we stained archival formalin-fixed, paraffin-embedded (FFPE) tissue from patients with primary prostate cancer in two prospective cohort studies, the Health Professionals Follow-up Study and the Physicians' Health Study. PTEN, stathmin, and phospho-S6 were quantified on 14 tissue microarrays as indicators of PI3K activation to derive cell-level PI3K scores.

Results: In 1,001 men, 988,254 tumor cells were assessed (median, 743 per tumor; interquartile range, 290-1,377). PI3K scores were higher in tumors with PTEN loss scored by a pathologist, higher Gleason grade, and a new, validated bulk PI3K transcriptional signature. Unsupervised machine-learning approaches resulted in similar clustering. Within-tumor heterogeneity in cell-level PI3K scores was high. During long-term follow-up (median, 15.3 years), rates of progression to metastases and death from prostate cancer were twice as high in the highest quartile of PI3K activation compared with the lowest quartile (hazard ratio, 2.04; 95% confidence interval, 1.13-3.68).

Conclusions: Our novel pathway-focused approach to quantifying single-cell-level immunofluorescence in FFPE tissue identifies prostate tumors with PI3K pathway activation that are more aggressive and may respond to pathway inhibitors.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7669556PMC
November 2020

Polyomavirus Small T Antigen Induces Apoptosis in Mammalian Cells through the UNC5B Pathway in a PP2A-Dependent Manner.

J Virol 2020 07 1;94(14). Epub 2020 Jul 1.

Department of Biochemistry, University of Kashmir, Srinagar, India

UNC5B is a dependence receptor that promotes survival in the presence of its ligand, netrin-1, while inducing cell death in its absence. The receptor has an important role in the development of the nervous and vascular systems. It is also involved in the normal turnover of intestinal epithelium. Netrin-1 and UNC5B are deregulated in multiple cancers, including colorectal, neuroblastoma, and breast tumors. However, the detailed mechanism of UNC5B function is not fully understood. We have utilized the murine polyomavirus small T antigen (PyST) as a tool to study UNC5B-mediated apoptosis. PyST is known to induce mitotic arrest followed by extensive cell death in mammalian cells. Our results show that the expression of PyST increases mRNA levels of UNC5B by approximately 3-fold in osteosarcoma cells (U2OS) and also stabilizes UNC5B at the posttranslational level. Furthermore, UNC5B is upregulated predominantly in those cells that undergo mitotic arrest upon PyST expression. Interestingly, although its expression was previously reported to be regulated by p53, our data show that the increase in UNC5B levels by PyST is p53 independent. The posttranslational stabilization of UNC5B by PyST is regulated by the interaction of PyST with PP2A. We also show that netrin-1 expression, which is known to inhibit UNC5B apoptotic activity, promotes survival of PyST-expressing cells. Our results thus suggest an important role of UNC5B in small-T antigen-induced mitotic catastrophe that also requires PP2A. UNC5B, PP2A, and netrin-1 are deregulated in a variety of cancers. UNC5B and PP2A are regarded as tumor suppressors, as they promote apoptosis and are deleted or mutated in many cancers. In contrast, netrin-1 promotes survival by inhibiting dependence receptors, including UNC5B, and is upregulated in many cancers. Here, we show that UNC5B-mediated apoptosis can occur independently of p53 but in a PP2A-dependent manner. A substantial percentage of cancers arise due to p53 mutations and are insensitive to chemotherapeutic treatments that activate p53. Unexpectedly, treatment of cancers having functional p53 with many conventional drugs leads to the upregulation of netrin-1 through activated p53, which is counterintuitive. Therefore, understanding the p53-independent mechanisms of the netrin-UNC5B axis, such as those involving PP2A, assumes greater clinical significance. Anticancer strategies utilizing anti-netrin-1 antibody treatment are already in clinical trials.
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http://dx.doi.org/10.1128/JVI.02187-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343204PMC
July 2020

Combination of KRAS gene silencing and PI3K inhibition for ovarian cancer treatment.

J Control Release 2020 02 12;318:98-108. Epub 2019 Dec 12.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02215, USA. Electronic address:

The phosphoinositide 3-kinase (PI3K) and RAS signaling pathways are frequently co-activated and altered during oncogenesis. Owing to their regulatory cross-talk, the early attempts of targeting only one pathway have mostly ended up promoting the development of drug resistance. Here, we propose using small interfering RNA (siRNA) therapeutics to directly target the undruggable KRAS (siKRAS) in combination with the pan-PI3K inhibitor GDC-0941 (GDC) to simultaneously block both PI3K and RAS signaling, thereby exerting synergistic anti-tumor effects on ovarian cancers with PTEN deficiency and KRAS mutation. For successful delivery of siKRAS, tGC/psi-nanoparticle formulation of polymerized siRNA and thiol-modified glycol chitosan nanoparticle-was used for KRAS specific inhibition in vitro and in vivo. GDC or siKRAS monotherapy each impede downstream signaling, leading to some delay in cell proliferation and migration. When combined, however, they engender much higher inhibition of PI3K signaling and stimulation of apoptosis in an ovarian allograft model compared to monotherapies. Our results show the feasibility of developing new combination strategies for the management of multiple oncogenic mutations activating PI3K and RAS signaling.
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http://dx.doi.org/10.1016/j.jconrel.2019.12.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056267PMC
February 2020

The Mechanisms Underlying PTEN Loss in Human Tumors Suggest Potential Therapeutic Opportunities.

Biomolecules 2019 11 7;9(11). Epub 2019 Nov 7.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

In this review, we will first briefly describe the diverse molecular mechanisms associated with PTEN loss of function in cancer. We will then proceed to discuss the molecular mechanisms linking PTEN loss to PI3K activation and demonstrate how these mechanisms suggest possible therapeutic approaches for patients with PTEN-null tumors.
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http://dx.doi.org/10.3390/biom9110713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6921025PMC
November 2019

Inhibition of the transcriptional kinase CDK7 overcomes therapeutic resistance in HER2-positive breast cancers.

Oncogene 2020 01 28;39(1):50-63. Epub 2019 Aug 28.

Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, 29425, USA.

Resistance of breast cancer to human epidermal growth factor receptor 2 (HER2) inhibitors involves reprogramming of the kinome through HER2/HER3 signaling via the activation of multiple tyrosine kinases and transcriptional upregulation. The heterogeneity of induced kinases prevents kinase targeting by a single kinase inhibitor and presents a major challenge to the treatment of therapeutically recalcitrant HER2-positive breast cancers (HER2+ BCs). As a result, there is a critical need for effective treatment that attacks the aberrant kinome activation associated with resistance to HER2-targeted therapy. Here, we describe a novel treatment strategy that targets cyclin-dependent kinase 7 (CDK7) in HER2 inhibitor-resistant (HER2iR) breast cancer. We show that both HER2 inhibitor-sensitive (HER2iS) and HER2iR breast cancer cell lines exhibit high sensitivity to THZ1, a newly identified covalent inhibitor of the transcription regulatory kinase CDK7. CDK7 promotes cell cycle progression through inhibition of transcription, rather than via direct phosphorylation of classical CDK targets. The transcriptional kinase activity of CDK7 is regulated by HER2, and by the receptor tyrosine kinases activated in response to HER2 inhibition, as well as by the downstream SHP2 and PI3K/AKT pathways. A low dose of THZ1 displayed potent synergy with the HER2 inhibitor lapatinib in HER2iR BC cells in vitro. Dual HER2 and CDK7 inhibition induced tumor regression in two HER2iR BC xenograft models in vivo. Our data support the utilization of CDK7 inhibition as an additional therapeutic avenue that blocks the activation of genes engaged by multiple HER2iR kinases.
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http://dx.doi.org/10.1038/s41388-019-0953-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6937212PMC
January 2020

Molecular Profiling Establishes Genetic Features Predictive of the Efficacy of the p110β Inhibitor KIN-193.

Cancer Res 2019 09 10;79(17):4524-4531. Epub 2019 Jul 10.

Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard Chan School of Public Health, Boston, Massachusetts.

Aberrant activation of the PI3K pathway is a common alteration in human cancers. Therapeutic intervention targeting the PI3K pathway has achieved limited success due to the intricate balance of its different components and isoforms. Here, we systematically investigated the genomic and transcriptomic signatures associated with response to KIN-193, a compound specifically targeting the p110β isoform. By integrating genomic, transcriptomic, and drug response profiles from the Genomics of Drug Sensitivity in Cancer database, we identified mutational and transcriptomic signatures associated with KIN-193 and further created statistical models to predict the treatment effect of KIN-193 in cell lines, which may eventually be clinically valuable. These predictions were validated by analysis of the external Cancer Cell Line Encyclopedia dataset. These results may assist precise therapeutic intervention targeting the PI3K pathway. SIGNIFICANCE: These findings provide new insights into molecular signatures associated with sensitivity of the p110β inhibitor KIN-193, which may provide a useful guide for developing precise treatment methods for cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-0588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726497PMC
September 2019

PI3 kinase alpha and delta promote hematopoietic stem cell activation.

JCI Insight 2019 05 23;5. Epub 2019 May 23.

Department of Medicine and.

Many cytokines and chemokines that are important for hematopoiesis activate the PI3K signaling pathway. Because this pathway is frequently mutated and activated in cancer, PI3K inhibitors have been developed for the treatment of several malignancies, and are now being tested in the clinic in combination with chemotherapy. However, the role of PI3K in adult hematopoietic stem cells (HSCs), particularly during hematopoietic stress, is still unclear. We previously showed that the individual PI3K catalytic isoforms P110α or P110β have dispensable roles in HSC function, suggesting redundancy between PI3K isoforms in HSCs. We now demonstrate that simultaneous deletion of P110α and P110δ in double knockout (DKO) HSCs uncovers their redundant requirement in HSC cycling after 5-fluorouracil (5-FU) chemotherapy administration. In contrast, DKO HSCs are still able to exit quiescence in response to other stress stimuli, such as LPS. We found that DKO HSCs and progenitors have impaired sensing of inflammatory signals ex vivo, and that levels of IL1-β and MIG are higher in the bone marrow after LPS than after 5-FU administration. Furthermore, exogenous in vivo administration of IL1-β can induce cell cycle entry of DKO HSCs. Our findings have important clinical implications for the use of PI3K inhibitors in combination with chemotherapy.
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http://dx.doi.org/10.1172/jci.insight.125832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629136PMC
May 2019

Buparlisib in Patients With Recurrent Glioblastoma Harboring Phosphatidylinositol 3-Kinase Pathway Activation: An Open-Label, Multicenter, Multi-Arm, Phase II Trial.

J Clin Oncol 2019 03 4;37(9):741-750. Epub 2019 Feb 4.

1 Dana-Farber Cancer Institute, Boston, MA.

Purpose: Phosphatidylinositol 3-kinase (PI3K) signaling is highly active in glioblastomas. We assessed pharmacokinetics, pharmacodynamics, and efficacy of the pan-PI3K inhibitor buparlisib in patients with recurrent glioblastoma with PI3K pathway activation.

Methods: This study was a multicenter, open-label, multi-arm, phase II trial in patients with PI3K pathway-activated glioblastoma at first or second recurrence. In cohort 1, patients scheduled for re-operation after progression received buparlisib for 7 to 13 days before surgery to evaluate brain penetration and modulation of the PI3K pathway in resected tumor tissue. In cohort 2, patients not eligible for re-operation received buparlisib until progression or unacceptable toxicity. Once daily oral buparlisib 100 mg was administered on a continuous 28-day schedule. Primary end points were PI3K pathway inhibition in tumor tissue and buparlisib pharmacokinetics in cohort 1 and 6-month progression-free survival (PFS6) in cohort 2.

Results: Sixty-five patients were treated (cohort 1, n = 15; cohort 2, n = 50). In cohort 1, reduction of phosphorylated AKT immunohistochemistry score was achieved in six (42.8%) of 14 patients, but effects on phosphoribosomal protein S6 and proliferation were not significant. Tumor-to-plasma drug level was 1.0. In cohort 2, four (8%) of 50 patients reached 6-month PFS6, and the median PFS was 1.7 months (95% CI, 1.4 to 1.8 months). The most common grade 3 or greater adverse events related to treatment were lipase elevation (n = 7 [10.8%]), fatigue (n = 4 [6.2%]), hyperglycemia (n = 3 [4.6%]), and elevated ALT (n = 3 [4.6%]).

Conclusion: Buparlisib had minimal single-agent efficacy in patients with PI3K-activated recurrent glioblastoma. Although buparlisib achieved significant brain penetration, the lack of clinical efficacy was explained by incomplete blockade of the PI3K pathway in tumor tissue. Integrative results suggest that additional study of PI3K inhibitors that achieve more-complete pathway inhibition may still be warranted.
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http://dx.doi.org/10.1200/JCO.18.01207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553812PMC
March 2019

PARP Inhibition Elicits STING-Dependent Antitumor Immunity in Brca1-Deficient Ovarian Cancer.

Cell Rep 2018 12;25(11):2972-2980.e5

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. Electronic address:

PARP inhibitors have shown promising clinical activities for patients with BRCA mutations and are changing the landscape of ovarian cancer treatment. However, the therapeutic mechanisms of action for PARP inhibition in the interaction of tumors with the tumor microenvironment and the host immune system remain unclear. We find that PARP inhibition by olaparib triggers robust local and systemic antitumor immunity involving both adaptive and innate immune responses through a STING-dependent antitumor immune response in mice bearing Brca1-deficient ovarian tumors. This effect is further augmented when olaparib is combined with PD-1 blockade. Our findings thus provide a molecular mechanism underlying antitumor activity by PARP inhibition and lay a foundation to improve therapeutic outcome for cancer patients.
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http://dx.doi.org/10.1016/j.celrep.2018.11.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366450PMC
December 2018

A Conditional Dependency on MELK for the Proliferation of Triple-Negative Breast Cancer Cells.

iScience 2018 Nov 18;9:149-160. Epub 2018 Oct 18.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

The role of maternal and embryonic leucine zipper kinase (MELK) in cancer cell proliferation has been contentious, with recent studies arriving at disparate conclusions. We investigated the in vitro dependency of cancer cells on MELK under a range of assay conditions. Abrogation of MELK expression has little effect under common culture conditions, in which cells are seeded at high densities and reach confluence in 3-5 days. However, MELK dependency becomes clearly apparent in clonogenic growth assays using either RNAi or CRISPR technologies to modulate MELK expression. This dependency is in sharp contrast to that of essential genes, such as those encoding classic mitotic kinases, but is similar to that of other oncogenes including MYC and KRAS. Our study provides an example demonstrating some of the challenges encountered in cancer target validation, and reveals how subtle, but important, technical variations can ultimately lead to divergent outcomes and conclusions.
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http://dx.doi.org/10.1016/j.isci.2018.10.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215964PMC
November 2018

Targeted Profiling of RNA Translation.

Curr Protoc Mol Biol 2019 01 22;125(1):e71. Epub 2018 Oct 22.

Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts.

This unit describes a reverse transcription-quantitative PCR (RT-qPCR)-based method for gene-targeted measurement of RNA translation levels. The method includes washing and lysing cells with a buffer containing cycloheximide to enrich ribosomal accumulation at translation initiation sites (TIS), followed by enzymatic treatment to generate ribosomal footprints, reverse transcription targeted towards TIS of specific transcripts of interest to generate complementary DNA (cDNA), and qPCR to measure the abundance of these footprints. This method enables time- and cost-effective assessment of changes in translation levels across focused panels of genes and across numerous samples. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmb.71DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326074PMC
January 2019

Targeted profiling of RNA translation reveals mTOR-4EBP1/2-independent translation regulation of mRNAs encoding ribosomal proteins.

Proc Natl Acad Sci U S A 2018 10 17;115(40):E9325-E9332. Epub 2018 Sep 17.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215;

The PI3K-Akt-mTOR signaling pathway is a master regulator of RNA translation. Pharmacological inhibition of this pathway preferentially and coordinately suppresses, in a 4EBP1/2-dependent manner, translation of mRNAs encoding ribosomal proteins. However, it is unclear whether mechanistic target of rapamycin (mTOR)-4EBP1/2 is the exclusive translation regulator of this group of genes, and furthermore, systematic searches for novel translation modulators have been immensely challenging because of difficulties in scaling existing RNA translation profiling assays. Here, we developed a rapid and highly scalable approach for gene-specific quantitation of RNA translation, termed Targeted Profiling of RNA Translation (TPRT). We applied this technique in a chemical screen for translation modulators, and identified numerous preclinical and clinical therapeutic compounds, with diverse nominal targets, that preferentially suppress translation of ribosomal proteins. Surprisingly, some of these compounds act in a manner that bypasses canonical regulation by mTOR-4EBP1/2. Instead, these compounds exert their translation effects in a manner that is dependent on GCN2-eIF2α, a central signaling axis within the integrated stress response. Furthermore, we were also able to identify metabolic perturbations that also suppress ribosomal protein translation in an mTOR-independent manner. Together, we describe a translation assay that is directly applicable to large-scale RNA translation studies, and that enabled us to identify a noncanonical, mTOR-independent mode for translation regulation of ribosomal proteins.
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http://dx.doi.org/10.1073/pnas.1805782115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176620PMC
October 2018

Isoform-Selective Phosphatidylinositol 3-Kinase Inhibition in Cancer.

J Clin Oncol 2018 05 8;36(13):1339-1342. Epub 2018 Mar 8.

Johann S. Bergholz, Thomas M. Roberts, and Jean J. Zhao, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA.

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http://dx.doi.org/10.1200/JCO.2017.77.0891DOI Listing
May 2018

CDK4/6 inhibition triggers anti-tumour immunity.

Nature 2017 08 16;548(7668):471-475. Epub 2017 Aug 16.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA.

Cyclin-dependent kinases 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle and are required for the initiation and progression of various malignancies. Pharmacological inhibitors of CDK4/6 have shown significant activity against several solid tumours. Their primary mechanism of action is thought to be the inhibition of phosphorylation of the retinoblastoma tumour suppressor, inducing G1 cell cycle arrest in tumour cells. Here we use mouse models of breast carcinoma and other solid tumours to show that selective CDK4/6 inhibitors not only induce tumour cell cycle arrest, but also promote anti-tumour immunity. We confirm this phenomenon through transcriptomic analysis of serial biopsies from a clinical trial of CDK4/6 inhibitor treatment for breast cancer. The enhanced anti-tumour immune response has two underpinnings. First, CDK4/6 inhibitors activate tumour cell expression of endogenous retroviral elements, thus increasing intracellular levels of double-stranded RNA. This in turn stimulates production of type III interferons and hence enhances tumour antigen presentation. Second, CDK4/6 inhibitors markedly suppress the proliferation of regulatory T cells. Mechanistically, the effects of CDK4/6 inhibitors both on tumour cells and on regulatory T cells are associated with reduced activity of the E2F target, DNA methyltransferase 1. Ultimately, these events promote cytotoxic T-cell-mediated clearance of tumour cells, which is further enhanced by the addition of immune checkpoint blockade. Our findings indicate that CDK4/6 inhibitors increase tumour immunogenicity and provide a rationale for new combination regimens comprising CDK4/6 inhibitors and immunotherapies as anti-cancer treatment.
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http://dx.doi.org/10.1038/nature23465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570667PMC
August 2017

CRKL Mediates p110β-Dependent PI3K Signaling in PTEN-Deficient Cancer Cells.

Cell Rep 2017 07;20(3):549-557

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02215, USA. Electronic address:

The p110β isoform of PI3K is preferentially activated in many tumors deficient in the phosphatase and tensin homolog (PTEN). However, the mechanism(s) linking PTEN loss to p110β activation remain(s) mysterious. Here, we identify CRKL as a member of the class of PI3Kβ-interacting proteins. Silencing CRKL expression in PTEN-null human cancer cells leads to a decrease in p110β-dependent PI3K signaling and cell proliferation. In contrast, CRKL depletion does not impair p110α-mediated signaling. Further study showed that CRKL binds to tyrosine-phosphorylated p130Cas in PTEN-null cancer cells. Since Src family kinases are known both to be regulated by PTEN and to phosphorylate and activate p130Cas, we tested and found that Src inhibition cooperated with p110β inhibition to suppress the growth of PTEN-null cells. These data suggest both a potential mechanism linking PTEN loss to p110β activation and the possible benefit of dual inhibition of Src and PI3K for PTEN-null tumors.
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http://dx.doi.org/10.1016/j.celrep.2017.06.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704918PMC
July 2017

PI3K-p110α mediates the oncogenic activity induced by loss of the novel tumor suppressor PI3K-p85α.

Proc Natl Acad Sci U S A 2017 07 19;114(27):7095-7100. Epub 2017 Jun 19.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115;

Mutation or loss of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is emerging as a transforming factor in cancer, but the mechanism of transformation has been controversial. Here we find that hemizygous deletion of the gene encoding p85α is a frequent event in breast cancer, with expression significantly reduced in breast tumors. knockdown transforms human mammary epithelial cells, and genetic ablation of accelerates a mouse model of HER2/neu-driven breast cancer. We demonstrate that partial loss of p85α increases the amount of p110α-p85 heterodimers bound to active receptors, augmenting PI3K signaling and oncogenic transformation. Pan-PI3K and p110α-selective pharmacological inhibition effectively blocks transformation driven by partial p85α loss both in vitro and in vivo. Together, our data suggest that p85α plays a tumor-suppressive role in transformation, and suggest that p110α-selective therapeutics may be effective in the treatment of breast cancer patients with loss.
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http://dx.doi.org/10.1073/pnas.1704706114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5502636PMC
July 2017

The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival.

Nature 2017 06 7;546(7658):426-430. Epub 2017 Jun 7.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA.

D-type cyclins (D1, D2 and D3) and their associated cyclin-dependent kinases (CDK4 and CDK6) are components of the core cell cycle machinery that drives cell proliferation. Inhibitors of CDK4 and CDK6 are currently being tested in clinical trials for patients with several cancer types, with promising results. Here, using human cancer cells and patient-derived xenografts in mice, we show that the cyclin D3-CDK6 kinase phosphorylates and inhibits the catalytic activity of two key enzymes in the glycolytic pathway, 6-phosphofructokinase and pyruvate kinase M2. This re-directs the glycolytic intermediates into the pentose phosphate (PPP) and serine pathways. Inhibition of cyclin D3-CDK6 in tumour cells reduces flow through the PPP and serine pathways, thereby depleting the antioxidants NADPH and glutathione. This, in turn, increases the levels of reactive oxygen species and causes apoptosis of tumour cells. The pro-survival function of cyclin D-associated kinase operates in tumours expressing high levels of cyclin D3-CDK6 complexes. We propose that measuring the levels of cyclin D3-CDK6 in human cancers might help to identify tumour subsets that undergo cell death and tumour regression upon inhibition of CDK4 and CDK6. Cyclin D3-CDK6, through its ability to link cell cycle and cell metabolism, represents a particularly powerful oncoprotein that affects cancer cells at several levels, and this property can be exploited for anti-cancer therapy.
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http://dx.doi.org/10.1038/nature22797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516959PMC
June 2017

Epigenetic regulation of RTK signaling.

J Mol Med (Berl) 2017 08 6;95(8):791-798. Epub 2017 Jun 6.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.

Receptor tyrosine kinase (RTK) signaling cascades coordinate intracellular signaling in response to growth factors, chemokines, and other extracellular stimuli to control fundamental biological processes such as cellular proliferation, metabolism, and survival. Hyperactivation of pathways associated with growth factor signaling (e.g., RTK and downstream effectors including Ras, PI3K/AKT, and Raf) is a frequent event in human cancers, which uncouples ligand-mediated activation with signal transduction. While the contributions of direct genomic events are well understood as causative agents of hyperactive signal transduction, other non-heritable genomic modifications promote the activation of growth factor-associated signaling cascades. In this review, we highlight epigenomic mechanisms by which hyperactivation of RTK-associated signaling cascades occurs and may contribute to cancer.
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http://dx.doi.org/10.1007/s00109-017-1546-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663182PMC
August 2017

The emerging role of PI3K/AKT-mediated epigenetic regulation in cancer.

Biochim Biophys Acta Rev Cancer 2017 Aug 14;1868(1):123-131. Epub 2017 Mar 14.

Department of Cancer Biology, Dana Farber Cancer Institute, 44 Binney St, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 44 Binney St, Boston, MA 02115, USA. Electronic address:

The PI3-kinase/AKT pathway integrates signals from external cellular stimuli to regulate essential cellular functions, and is frequently aberrantly activated in human cancers. Recent research demonstrates that tight regulation of the epigenome is critical in preserving and restricting transcriptional activation, which can impact cellular growth and proliferation. In this review we examine mechanisms by which the PI3K/AKT pathway regulates the epigenome to promote oncogenesis, and highlight how connections between PI3K/AKT and the epigenome may impact the future therapeutic treatment of cancers featuring a hyperactivated PI3K/AKT pathway.
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http://dx.doi.org/10.1016/j.bbcan.2017.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548615PMC
August 2017

Rac1-mediated membrane raft localization of PI3K/p110β is required for its activation by GPCRs or PTEN loss.

Elife 2016 10 4;5. Epub 2016 Oct 4.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, United States.

We aimed to understand how spatial compartmentalization in the plasma membrane might contribute to the functions of the ubiquitous class IA phosphoinositide 3-kinase (PI3K) isoforms, p110α and p110β. We found that p110β localizes to membrane rafts in a Rac1-dependent manner. This localization potentiates Akt activation by G-protein-coupled receptors (GPCRs). Thus genetic targeting of a Rac1 binding-deficient allele of p110β to rafts alleviated the requirement for p110β-Rac1 association for GPCR signaling, cell growth and migration. In contrast, p110α, which does not play a physiological role in GPCR signaling, is found to reside in nonraft regions of the plasma membrane. Raft targeting of p110α allowed its EGFR-mediated activation by GPCRs. Notably, p110β dependent, PTEN null tumor cells critically rely upon raft-associated PI3K activity. Collectively, our findings provide a mechanistic account of how membrane raft localization regulates differential activation of distinct PI3K isoforms and offer insight into why PTEN-deficient cancers depend on p110β.
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http://dx.doi.org/10.7554/eLife.17635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5050018PMC
October 2016

NTRK2 activation cooperates with PTEN deficiency in T-ALL through activation of both the PI3K-AKT and JAK-STAT3 pathways.

Cell Discov 2016 20;2:16030. Epub 2016 Sep 20.

Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.

Loss of PTEN, a negative regulator of the phosphoinositide 3-kinase signaling pathway, is a frequent event in T-cell acute lymphoblastic leukemia, suggesting the importance of phosphoinositide 3-kinase activity in this disease. Indeed, hyperactivation of the phosphoinositide 3-kinase pathway is associated with the disease aggressiveness, poor prognosis and resistance to current therapies. To identify a molecular pathway capable of cooperating with PTEN deficiency to drive oncogenic transformation of leukocytes, we performed an unbiased transformation screen with a library of tyrosine kinases. We found that activation of NTRK2 is able to confer a full growth phenotype of Ba/F3 cells in an IL3-independent manner in the PTEN-null setting. NTRK2 activation cooperates with PTEN deficiency through engaging both phosphoinositide3-kinase/AKT and JAK/STAT3 pathway activation in leukocytes. Notably, pharmacological inhibition demonstrated that p110α and p110δ are the major isoforms mediating the phosphoinositide 3-kinase/AKT signaling driven by NTRK2 activation in PTEN-deficient leukemia cells. Furthermore, combined inhibition of phosphoinositide 3-kinase and STAT3 significantly suppressed proliferation of PTEN-mutant T-cell acute lymphoblastic leukemia both in culture and in mouse xenografts. Together, our data suggest that a unique conjunction of PTEN deficiency and NTRK2 activation in T-cell acute lymphoblastic leukemia, and combined pharmacologic inhibition of phosphoinositide 3-kinase and STAT3 signaling may serve as an effective and durable therapeutic strategy for T-cell acute lymphoblastic leukemia.
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http://dx.doi.org/10.1038/celldisc.2016.30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5029543PMC
September 2016

Combined inhibition of PI3K and PARP is effective in the treatment of ovarian cancer cells with wild-type PIK3CA genes.

Gynecol Oncol 2016 09 15;142(3):548-56. Epub 2016 Jul 15.

Cancer Institute, The Second Hospital of Dalian Medical University, Institute of Cancer Stem Cell, Dalian Medical University, Dalian 116044, China. Electronic address:

Objective: Combined inhibition of PI3K and PARP has been shown to be effective in the treatment of preclinical models of breast cancer and prostate cancer independent of BRCA or PIK3CA mutational status. However, the knowledge about this combination treatment in ovarian cancer is limited. The aim of this study was to evaluate the therapeutic effect of PI3K inhibitor BKM120 and PARP inhibitor Olaparib on ovarian cancer cell lines bearing wild-type PIK3CA genes.

Methods: We exposed three wild-type PIK3CA ovarian cancer cell lines to a PI3K inhibitor BKM120 and/or a PARP inhibitor Olaparib. The effect of BKM120 as a single-agent or in combination with Olaparib was evaluated by Cell Count Kit (CCK8) assay, immunoblotting, comet assay, flow cytometry and immunofluorescence staining assay. The combination indexes for synergistic effect on cell viability were calculated with the Chou-Talalay method. Ex vivo cultured ovarian cancer tissues from patients were analyzed by histological and immunohistochemical analyses.

Results: Combined inhibition of PI3K and PARP effectively synergized to block the growth of three wild-type PIK3CA ovarian cancer cell lines and explants of a primary ovarian tumor specimen. Mechanistically, dual blockade of PI3K and PARP in these ovarian cancer cell lines resulted in substantially attenuated PI3K/AKT/mTOR signaling, impaired DNA damage response and deficient homologous recombination repair, with remarkable BRCA downregulation.

Conclusions: The combined use of PI3K inhibitor BKM120 and PARP inhibitor Olaparib may be effective in ovarian cancers with a broader spectrum of cancer-associated genetic alterations but not limited to those with mutant PIK3CA or BRCA genes. BRCA downregulation may be a potential biomarker for the effective response to the proposed combination treatment.
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http://dx.doi.org/10.1016/j.ygyno.2016.07.092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551048PMC
September 2016

Tyrosine receptor kinase B is a drug target in astrocytomas.

Neuro Oncol 2017 01 10;19(1):22-30. Epub 2016 Jul 10.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts (J.N., S.X., V.L., Y.S., P.B., R.B., T.M.R., C.D.S., R.A.S., J.J.Z.); Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts (J.N., S.X., V.L., T.M.R., J.J.Z.); Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts (S.H.R., R.B., K.L.L., W.C.H.); Broad Institute, Boston, Massachusetts (P.B., R.B., W.C.H.); Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts (S.H.R., K.L.L.); Department of Pathology, Boston Children's Hospital, Boston, Massachusetts (K.L.L.)

Background: Astrocytomas are the most common primary human brain tumors. Receptor tyrosine kinases (RTKs), including tyrosine receptor kinase B (TrkB, also known as tropomyosin-related kinase B; encoded by neurotrophic tyrosine kinase receptor type 2 [NTRK2]), are frequently mutated by rearrangement/fusion in high-grade and low-grade astrocytomas. We found that activated TrkB can contribute to the development of astrocytoma and might serve as a therapeutic target in this tumor type.

Methods: To identify RTKs capable of inducing astrocytoma formation, a library of human tyrosine kinases was screened for the ability to transform murine Ink4a/Arf astrocytes. Orthotopic allograft studies were conducted to evaluate the effects of RTKs on the development of astrocytoma. Since TrkB was identified as a driver of astrocytoma formation, the effect of the Trk inhibitors AZD1480 and RXDX-101 was assessed in astrocytoma cells expressing activated TrkB. RNA sequencing, real-time PCR, western blotting, and enzyme-linked immunosorbent assays were conducted to characterize NTRK2 in astrocytomas.

Results: Activated TrkB cooperated with Ink4a/Arf loss to induce the formation of astrocytomas through a mechanism mediated by activation of signal transducer and activator of transcription 3 (STAT3). TrkB activation positively correlated with Ccl2 expression. TrkB-induced astrocytomas remained dependent on TrkB signaling for survival, highlighting a role of NTRK2 as an addictive oncogene. Furthermore, the QKI-NTRK2 fusion associated with human astrocytoma transformed Ink4a/Arf astrocytes, and this process was also mediated via STAT3 signaling.

Conclusions: Our findings provide evidence that constitutively activated NTRK2 alleles, notably the human tumor-associated QKI-NTRK2 fusion, can cooperate with Ink4a/Arf loss to drive astrocytoma formation. Therefore, we propose NTRK2 as a potential therapeutic target in the subset of astrocytoma patients defined by QKI-NTRK2 fusion.
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http://dx.doi.org/10.1093/neuonc/now139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5193024PMC
January 2017
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