Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits SMARCA4 and ...SMARCA2. A great need exists for effective targeted therapies for SCCOHT.
To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT.
The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%.
Ponatinib is an effective agent for
-mutant SCCOHT in both
and
preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted.
.
We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of ...melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
There is substantial evidence that in addition to nicotine, other compounds found in tobacco smoke significantly influence smoking behavior. Further, recent years have seen an explosion in the ...availability of non-combusted products that deliver nicotine, such as e-cigarettes and “home-brew” vaping devices that are essentially unregulated. There are many thousands of compounds in tobacco smoke alone, and new products are constantly introducing new compounds. Uncovering which of these compounds are active, across multiple smoking-relevant subtypes of the nicotinic acetylcholine receptor (nAChR) that influence tobacco/nicotine addiction, requires a high-throughput screening (HTS) approach. Accordingly, we developed a panel of HTS-friendly cell-based assays, all performed in the same cellular background and using the same membrane potential dye readout, to measure the function of the α3β4-, α4β2-, and α6β2-nAChR subtypes. These subtypes have each been prominently and consistently associated with human smoking behavior. We validated our assays by performing pilot screening of an expanded set of the Prestwick FDA-approved drug library. The screens displayed excellent performance parameters, and moderate hit rates (mean of 1.2% across all three assays) were achieved when identifying antagonists (chosen since effects of endogenous antagonists on consumption of nicotine/tobacco products are under-studied). Validation rates using an orthogonal assay (86Rb+ efflux) averaged 73% across the three assays. The resulting panel of assays represents a valuable new platform with which to screen and identify nAChR subtype-selective compounds. This provides a resource for identifying smoking-related compounds in both combusted and non-combusted tobacco products, with potential relevance in the search for additional smoking-cessation therapies.
Glioblastoma (GB) is the most lethal brain cancer in adults, with a 5-year survival rate of 5%. The standard of care for GB includes maximally safe surgical resection, radiation, and temozolomide ...(TMZ) therapy, but tumor recurrence is inevitable in most GB patients. Here, we describe the development of a blood-brain barrier (BBB)-penetrant tubulin destabilizer, RGN3067, for the treatment of GB. RGN3067 shows good oral bioavailability and achieves high concentrations in rodent brains after oral dosing (C
of 7807 ng/mL (20 μM), T
at 2 h). RGN3067 binds the colchicine binding site of tubulin and inhibits tubulin polymerization. The compound also suppresses the proliferation of the GB cell lines U87 and LN-18, with IC
s of 117 and 560 nM, respectively. In four patient-derived GB cell lines, the IC
values for RGN3067 range from 148 to 616 nM. Finally, in a patient-derived xenograft (PDX) mouse model, RGN3067 reduces the rate of tumor growth compared to the control. Collectively, we show that RGN3067 is a BBB-penetrant small molecule that shows in vitro and in vivo efficacy and that its design addresses many of the physicochemical properties that prevent the use of microtubule destabilizers as treatments for GB and other brain cancers.
Abstract
Advancing new therapeutics to FDA approval for GBM is hampered by intra- and inter-tumoral heterogeneity. Design of clinical trials to enroll patients likely to respond to a new treatment ...seems critical to build the arsenal of agents against this disease. Neddylation is a protein modification pathway aligned to protein degradation and cytological localization within the ubiquitin/proteasome system. Components of the neddylation pathway are frequently overexpressed or hyperactivated in GBM, and whose upregulation has been associated with glioma progression and worse survival. Among glioblastoma cell lines and patient-derived stem cells, subsets were found to be selectively susceptible to NAE inhibitors. We previously showed that PTEN signaling, DNA replication, and chromatin instability pathways are the most significant differentiators between MLN4924 sensitive vs. non-sensitive models. To test a candidate molecular “signature of vulnerability”, two subsets of PDX models (PTEN wildtype and PTEN del or mutated) were treated with a panel of 3 NAE inhibitors. 5 GBM PTENwt models were shown vulnerable to MLN4924 and TAS4464 with average EC50 values 5-10 folds lower than 6 PTENmt/del models. CDC activity was greater against PTENwt, but only 2-fold different vs PTENmt/del. Transcriptomic data mining uncovered additional determinants of response to NAE inhibitors. To disclose the relationship between constitutive expression of three gene sets: DNA Replication Fork Progressing, Replication Fork Protection, and Replication Fork Protection Complex, we found that the genes involved in these are significantly under expressed in PTENmt models when comparing to PTENwt models. The molecular determinants of drug response to NAE inhibitors will be further verified in the extended preclinical models and lead to a patient-enrollment “signature of vulnerability” to increase the likelihood of demonstrating therapeutic efficacy in the early stage of clinical trials.
Pancreatic ductal adenocarcinoma (PDA) is a highly lethal cancer characterized by complex aberrant genomes. A fundamental goal of current studies is to identify those somatic events arising in the ...variable landscape of PDA genomes that can be exploited for improved clinical outcomes.
We used DNA content flow sorting to identify and purify tumor nuclei of PDA samples from 50 patients. The genome of each sorted sample was profiled by oligonucleotide comparative genomic hybridization and targeted resequencing of STAG2. Transposon insertions within STAG2 in a KRAS (G12D)-driven genetically engineered mouse model of PDA were screened by RT-PCR. We then used a tissue microarray to survey STAG2 protein expression levels in 344 human PDA tumor samples and adjacent tissues. Univariate Kaplan Meier analysis and multivariate Cox Regression analysis were used to assess the association of STAG2 expression relative to overall survival and response to adjuvant therapy. Finally, RNAi-based assays with PDA cell lines were used to assess the potential therapeutic consequence of STAG2 expression in response to 18 therapeutic agents.
STAG2 is targeted by somatic aberrations in a subset (4%) of human PDAs. Transposon-mediated disruption of STAG2 in a KRAS (G12D) genetically engineered mouse model promotes the development of PDA and its progression to metastatic disease. There was a statistically significant loss of STAG2 protein expression in human tumor tissue (Wilcoxon-Rank test) with complete absence of STAG2 staining observed in 15 (4.3%) patients. In univariate Kaplan Meier analysis nearly complete STAG2 positive staining (>95% of nuclei positive) was associated with a median survival benefit of 6.41 months (P = 0.031). The survival benefit of adjuvant chemotherapy was only seen in patients with a STAG2 staining of less than 95% (median survival benefit 7.65 months; P = 0.028). Multivariate Cox Regression analysis showed that STAG2 is an independent prognostic factor for survival in pancreatic cancer patients. Finally, we show that RNAi-mediated knockdown of STAG2 selectively sensitizes human PDA cell lines to platinum-based therapy.
Based on these iterative findings we propose that STAG2 is a clinically significant tumor suppressor in PDA.
Abstract
Glioblastoma (GBM), a particularly aggressive form of primary brain tumor, has poor survival due to a lack of effective treatments and high recurrence. Epidermal growth factor receptor ...(EGFR) (mapped to 7p 11.2), is frequently amplified and mutated (>50%) in GBM, and is suggested as a potential therapeutic target. To correlate chemo-vulnerability with genomic aberrations and further determine biomarkers for targeted therapies, a panel of seven EGFR tyrosine kinase inhibitors were tested in a selected nine human GBM patient-derived xenograft (PDX) panel; these preclinical models harbor different mutation combinations of CDKN2A deletion (C), PTEN deletion (P), wild-type EGFR (E) and/or EGFRvIII (Ev3) overexpression. A single agent drug dose response assay (DDR) was performed on GBM PDX neurospheres using drug concentrations ranging from 100uM to 0.5nM with a 12-point serial 3-fold dilution scheme. The cytotoxic efficacy of EGFR inhibition was dependent on both the PDX line and the EFGR inhibitor applied. GBM59 and GBM76 – both triple CEv3P mutants – were the most responsive to EGFR inhibition relative to the other GBM models tested. For GBM59, the IC50 values of afatinib, canertinib and neratinib, were sub-micromolar, and low micromolar (<3uM) for erlotinib, gefitinib and lapatinib. GBM76 had IC50 values of 1-8uM with all seven EGFR inhibitors including AZD3759. However, GBM155 and GBM126 with CEv3 but wildtype PTEN only responded to afatinib, canertinib and neratinib at low micromolar IC50 values (<3uM) but not to erlotinib, gefitinib and lapatinib. A deletion in PTEN may sensitize GBM cells with CEv3 mutations to erlotinib, gefitinib and lapatinib in vitro. The other group of GBM PDX lines (C only) represented by GBM122, GBM150 and GBM182 responded well to afatinib, canertinib and neratinib with IC50 values of 2- 9uM. GBM156 with CE had a similar profile to the group of C only or CEv3. GBM56 with CP showed the most resistance with mild responses to afatinib, canertinib and neratinib, as observed in its high micromolar IC50 values (6-9uM). Overall, neratinib is the most effective compound among the tested EGFR inhibitors based on IC50 values. Canertinib, the triple tyrosine kinase inhibitor against EGFR/HER2/ErbB-4, is the second most effective agent. Afatinib has similar drug efficacy to canertinib. All three compounds moderately inhibit all GBM PDX lines. The drug response mechanism and its correlation with the mutation status of CDKN2A, EGFR, and PTEN will be further investigated cross a broad array of GBM PDX models. Supported by NIH NCI R01 CA204136
Citation Format: Nanyun Tang, George Reid, C Ryan Miller, Michael E. Berens. Selective vulnerability of GBM PDX to a panel of EGFR tyrosine kinase inhibitors abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3681.
Myo1b is a widely expressed myosin-I isoform that concentrates on endosomal and ruffling membranes and is thought to play roles in membrane trafficking and dynamics. Myo1b is alternatively spliced ...within the regulatory domain of the molecule, yielding isoforms with six (myo1ba), five (myo1bb), or four (myo1bc) non-identical IQ motifs. The calmodulin binding properties of the myo1b IQ motifs have not been investigated, and the mechanical and cell biological consequences of alternative splicing are not known. Therefore, we expressed the alternatively spliced myo1b isoforms truncated after the final IQ motif and included a sequence at their C termini that is a substrate for bacterial biotin ligase. Site-specific biotinylation allows us to specifically attach the myosin to motility surfaces via a biotin-streptavidin linkage. We measured the ATPase and motile properties of the recombinant myo1b splice isoforms, and we correlated these properties with calmodulin binding. We confirmed that calcium-dependent changes in the ATPase activity are due to calcium binding to the calmodulin closest to the motor. We found that calmodulin binds tightly to some of the IQ motifs (Kd < 0.2 μm) and very weakly to the others (Kd > 5 μm), suggesting that a subset of the IQ motifs are not calmodulin bound under physiological conditions. Finally, we found the in vitro motility rate to be dependent on the myo1b isoform and the calmodulin concentration and that the myo1b regulatory domain acts as a rigid lever arm upon calmodulin binding to the high affinity and low affinity IQ motifs.
Abstract
Cell-free DNA (cfDNA) are short fragments of DNA that are released by apoptotic or necrotic cells into bodily fluids. It has emerged as a potential diagnostic tool allowing for the detection ...and analysis of tumor-derived biomarkers in cancer. Despite the detailed view provided by clinical imaging, its inability to quantify tumor burden and distinguish between pseudo-progression and pseudo-response poses a challenge for patients. In this study, we sought to investigate the effectiveness of cfDNA as a liquid biopsy tool to track tumor burden and disease progression in real time in different glioblastoma models and through different types of biofluids. To examine the feasibility of cfDNA-based tumor tracking we cultivated several patient-derived glioblastoma lines, purified cfDNA and performed sequencing. Further analysis revealed that dead cells were associated with an elevated cfDNA yield and altered fragment size profiles. Genomic sequencing revealed consistent variants shared across serial cfDNA timepoints, strengthening the suggestion that cfDNA can be used as a non-invasive biomarker. In order to demonstrate the clinical implications of our methodology, we collected a preliminary batch of n = 43 longitudinal intraventricular cerebrospinal fluid (CSF) samples from a patient that underwent CAR-T treatment over the span of two years. We observed a marked increase in cfDNA yield post-treatment compared to samples taken prior to treatment administration. Furthermore, cfDNA-based estimates of tumor burden measured as allele fraction specific to the tumor, tracked with clinical imaging-based tumor size estimates. These preliminary findings provide compelling evidence supporting the clinical utility of cfDNA analysis in monitoring the effectiveness and dynamics of CAR-T treatment. By tracking cfDNA levels in CSF, we can identify patterns reflecting treatment response and aid in assessing the therapeutic efficiency of CAR-T treatment in glioma patients. Further investigations with larger patient cohorts and more comprehensive data analysis to validate these findings are actively underway.
Abstract
Using two H3K27M-DMG treatment-naive preclinical models (PBT22 and PBT29), we detected a 20-fold differential response to the histone deacetylase inhibitor, Quisinostat. PBT-22 harbors ...mutations in H3F3A, TP53, and ASXL2, while PBT-29 has mutations in H3F3A, TP53, PIK3CA and FGFR1. Acetylation and deacetylation of wtH3 alter chromatin structure as part of normal regulation of gene expression. The methionine substitution for Lysine in H3K27M removes this histone from participating in acetylation/deacetylation regulated gene expression. Concomitantly, H3K27M induces a marked reduction in global acetylation of histone tails on wtH3. In the native state of H3K27M DMG with hypoacetylated H3, we posit that the specific pattern of nucleosome integration with genes responsible for cell survival/death underlies the differential vulnerability of DMGs to HDACi. Additionally, the nucleosome integration pattern with cell survival/death genes may be based on the relative abundance of wtH3wt versus H3K27M- histones. Following Quis treatment (48 hrs) in both preclinical models, total wtH3ac protein abundance increased 3-fold, suggesting HDACi stabilizes or impedes K27 acetylated H3 histone turnover. Comparison of differentially expressed genes (DEGs) from control and Quis-treated PBT22 and PBT29 will seed gene ontology analysis focusing on chromatin remodeling, cell death, and growth arrest pathways accounting for differential vulnerability. Findings will be validated by measuring transcripts and proteins (qRT-PCR and ELISA or mass spectrometry) from analytes from a larger panel of DMG cell lines. The data depicts DMG preclinical models with large differential sensitivity to Quis. The long-term goal is to discover a molecular profile of DMGs predictive of the vulnerability to HDACi. The discovery exercise is also likely to yield insight into the development of resistance to HDAC inhibitors.
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