[Show abstract][Hide abstract] ABSTRACT: Significance:
This study adds to the growing evidence that oncogenic pathways in tumors can promote resistance to the anti-tumor immune response. As PTEN loss and PI3K-AKT pathway activation occur in multiple tumor types, the results support the rationale to further evaluate combinatorial strategies targeting the PI3K-AKT pathway to increase the efficacy of immunotherapy.
[Show abstract][Hide abstract] ABSTRACT: Background:
Proteins that 'read' the histone code are central elements in epigenetic control and bromodomains, which bind acetyl-lysine motifs, are increasingly recognized as potential mediators of disease states. Notably, the first BET bromodomain-based therapies have entered clinical trials and there is a broad interest in dissecting the therapeutic relevance of other bromodomain-containing proteins in human disease. Typically, drug development is facilitated and expedited by high-throughput screening, where assays need to be sensitive, robust, cost-effective and scalable. However, for bromodomains, which lack catalytic activity that otherwise can be monitored (using classical enzymology), the development of cell-based, drug-target engagement assays has been challenging. Consequently, cell biochemical assays have lagged behind compared to other protein families (e.g., histone deacetylases and methyltransferases).
Here, we present a suite of novel chromatin and histone-binding assays using AlphaLISA, in situ cell extraction and fluorescence-based, high-content imaging. First, using TRIM24 as an example, the homogenous, bead-based AlphaScreen technology was modified from a biochemical peptide-competition assay to measure binding of the TRIM24 bromodomain to endogenous histone H3 in cells (AlphaLISA). Second, a target agnostic, high-throughput imaging platform was developed to quantify the ability of chemical probes to dissociate endogenous proteins from chromatin/nuclear structures. While overall nuclear morphology is maintained, the procedure extracts soluble, non-chromatin-bound proteins from cells with drug-target displacement visualized by immunofluorescence (IF) or microscopy of fluorescent proteins. Pharmacological evaluation of these assays cross-validated their utility, sensitivity and robustness. Finally, using genetic and pharmacological approaches, we dissect domain contribution of TRIM24, BRD4, ATAD2 and SMARCA2 to chromatin binding illustrating the versatility/utility of the in situ cell extraction platform.
In summary, we have developed two novel complementary and cell-based drug-target engagement assays, expanding the repertoire of pharmacodynamic assays for bromodomain tool compound development. These assays have been validated through a successful TRIM24 bromodomain inhibitor program, where a micromolar lead molecule (IACS-6558) was optimized using cell-based assays to yield the first single-digit nanomolar TRIM24 inhibitor (IACS-9571). Altogether, the assay platforms described herein are poised to accelerate the discovery and development of novel chemical probes to deliver on the promise of epigenetic-based therapies.
[Show abstract][Hide abstract] ABSTRACT: Glioma sphere-forming cells (GSCs) play important roles in tumor maintenance and chemotherapy and radiation resistance, and
thus serve as important models for the development of novel therapies and for understanding tumor biology. We hypothesized
that genomic analyses of GSCs in the absence of normal stroma would identify novel somatic alterations not previously reported
from whole tumor specimen analyses. We analyzed 42 GSCs, derived at the time of surgical resection, and their matched tumors
of origin. Unsupervised consensus clustering by non-negative matrix factorization (CNMF) using whole transcriptome sequencing
data identified 5 transcriptional classes. Comparison to published TCGA GBM subtypes using single-sample gene set enrichment
analysis (ssGSEA) showed that 9 GSCs perfectly matched with TCGA mesenchymal subtype. The other 4 subgroups of GSCs mapped
to proneural and/or classical subtypes, while none of the GSC classes matched with the neural TCGA subtype. Overall, 73% of
GSCs matched the TCGA subtype of paried tumor, suggesting transcriptional consistency between GSCs and their parental tumor.
A total of 88 candidate gene fusions were also identified in the GSCs, including the previously reported FGFR3-TACC3 and EGFR-SEP14
fusions. DNA copy number analysis indicated recurrent regions of frequent loss or gain, such as EGFR, MYC, and MYCN amplification,
and deletion of PTEN and CDKN2A. Whole exome sequencing based analysis identified 3,918 somatic mutations in 22 GSCs. PTEN,
TP53, EGFR and 17 additional genes were identified as significantly altered. Our results indicate genomic consistency between
GSCs and their tumors of origin, suggesting the suitability of GSCs as a model for preclinical testing. Additional gene fusions
and somatic mutations were detected in GSCs suggesting the important role of these cell models in identifying infrequent events
only detectable in pure tumor cell populations.
[Show abstract][Hide abstract] ABSTRACT: The mesenchymal (Mes) subtype of glioblastoma (MesGBM) correlates with poor patient prognosis. Surprisingly, agents that target
mesenchymal tumors, such as the antiangiogenesis drug bevacizumab, failed to show a benefit in MesGBM. The expression of MesGBM
might result from both cells from the surrounding microenvironment or from the tumor cells themselves. We hypothesized that
MesGBM driven by Mes-expressing tumor cells would show increased aggressiveness compared to those driven by Mes-expressing
stroma. We examined 42 glioma sphere-forming cell (GSCs) lines, derived from fresh surgical specimens, by performing whole
transcriptome sequencing, copy number analysis, and whole exome sequencing and compared the GSCs to the tumors from which
they were derived. GSCs aggressiveness was determined by in vivo survival of orthotopic xenografted mice and in vitro radiation
survival. The TCGA expression subtype of the GSCs and their matched tumor was determined by single sample gene set enrichment
analysis (ssGSEA). Of the 15 tumors belonging to the mesenchymal subtype, 4 of the matched GSCs were also mesenchymal (concordant
pairs, CP) and the remaining 11 GSCs were either proneural or classical subtypes (discordant pairs, DP). The normalized mesenchymal
scores of the 4 CP tumors (mean 0.90, range 0.80-0.98) was higher than those the 11 DP tumors (mean 0.75, range 0.69-0.90).
We determined tumor purity based on sequencing data and found it significantly higher in DP tumors than CP, suggesting a stromal
contribution to the Mes signature in the DP tumors. In addition, EGFR amplification occurred in 36% of DP tumors, and 0% of
TP tumors. CP tumors showed increased radiation resistance and decreased in vivo survival compared to DP tumors. In summary,
stromal-driven mesenchymal tumors showed decrease aggressiveness compared to tumor cell-driven MesGBM, suggesting that therapeutics
targeting the microenvironment, such as anti-angiogenesis drugs, may have a greater role in tumors where the stromal contribution
[Show abstract][Hide abstract] ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in western countries, with a median survival of 6 months and an extremely low percentage of long-term surviving patients. KRAS mutations are known to be a driver event of PDAC, but targeting mutant KRAS has proved challenging. Targeting oncogene-driven signalling pathways is a clinically validated approach for several devastating diseases. Still, despite marked tumour shrinkage, the frequency of relapse indicates that a fraction of tumour cells survives shut down of oncogenic signalling. Here we explore the role of mutant KRAS in PDAC maintenance using a recently developed inducible mouse model of mutated Kras (Kras(G12D), herein KRas) in a p53(LoxP/WT) background. We demonstrate that a subpopulation of dormant tumour cells surviving oncogene ablation (surviving cells) and responsible for tumour relapse has features of cancer stem cells and relies on oxidative phosphorylation for survival. Transcriptomic and metabolic analyses of surviving cells reveal prominent expression of genes governing mitochondrial function, autophagy and lysosome activity, as well as a strong reliance on mitochondrial respiration and a decreased dependence on glycolysis for cellular energetics. Accordingly, surviving cells show high sensitivity to oxidative phosphorylation inhibitors, which can inhibit tumour recurrence. Our integrated analyses illuminate a therapeutic strategy of combined targeting of the KRAS pathway and mitochondrial respiration to manage pancreatic cancer.
[Show abstract][Hide abstract] ABSTRACT: Glioma sphere-forming cells (GSCs) derived from surgical specimens are a fundamental resource to study glioblastoma (GBM) biology. Mesenchymal-expressing GSCs have been proposed as a source of treatment resistance and mesenchymal tumors correlate with poorer survival. Recently, we found that the anti-angiogensis drug bevacizumab appeared to provide no benefit to patients with mesenchymal tumors, in contradiction to expectations that a mesenchymal microenvironment may benefit from anti-angiogenesis therapy. We have developed a collection of GSCs that have undergone comprehensive genomic characterization, similar to that performed by the Cancer Genome Atlas (TCGA) for whole tumor specimens. We hypothesized that the genomic landscape of GSCs would recapitulate what was observed by TCGA.
[Show abstract][Hide abstract] ABSTRACT: Activating mutations in KRAS are among the most frequent events in diverse human carcinomas and are particularly prominent in human pancreatic ductal adenocarcinoma (PDAC). An inducible Kras(G12D)-driven mouse model of PDAC has established a critical role for sustained Kras(G12D) expression in tumor maintenance, providing a model to determine the potential for and the underlying mechanisms of Kras(G12D)-independent PDAC recurrence. Here, we show that some tumors undergo spontaneous relapse and are devoid of Kras(G12D) expression and downstream canonical MAPK signaling and instead acquire amplification and overexpression of the transcriptional coactivator Yap1. Functional studies established the role of Yap1 and the transcriptional factor Tead2 in driving Kras(G12D)-independent tumor maintenance. The Yap1/Tead2 complex acts cooperatively with E2F transcription factors to activate a cell cycle and DNA replication program. Our studies, along with corroborating evidence from human PDAC models, portend a novel mechanism of escape from oncogenic Kras addiction in PDAC.
[Show abstract][Hide abstract] ABSTRACT: Melanoma is a highly lethal malignancy notorious for its aggressive clinical course and eventual resistance to existing therapies. Currently, we possess a limited understanding of the genetic events driving melanoma progression, and much effort is focused on identifying pro-metastatic aberrations or perturbed signaling networks that constitute new therapeutic targets. In this study, we validate and assess the mechanism by which homeobox transcription factor A1 (HOXA1), a pro-invasion oncogene previously identified in a metastasis screen by our group, contributes to melanoma progression. Transcriptome and pathway profiling analyses of cells expressing HOXA1 reveals upregulation of factors involved in diverse cytokine pathways that include the transforming growth factor beta (TGFβ) signaling axis, which we further demonstrate to be required for HOXA1-mediated cell invasion in melanoma cells. Transcriptome profiling also shows HOXA1's ability to potently downregulate expression of microphthalmia-associated transcription factor (MITF) and other genes required for melanocyte differentiation, suggesting a mechanism by which HOXA1 expression de-differentiates cells into a pro-invasive cell state concomitant with TGFβ activation. Our analysis of publicly available data sets indicate that the HOXA1-induced gene signature successfully categorizes melanoma specimens based on their metastatic potential and, importantly, is capable of stratifying melanoma patient risk for metastasis based on expression in primary tumors. Together, these validation data and mechanistic insights suggest that patients whose primary tumors express HOXA1 are among a high-risk metastasis subgroup that should be considered for anti-TGFβ therapy in adjuvant settings. Moreover, further analysis of HOXA1 target genes in melanoma may reveal new pathways or targets amenable to therapeutic intervention.Oncogene advance online publication, 25 February 2013; doi:10.1038/onc.2013.30.