FOLFIRINOX, a combination of chemotherapy drugs (Fluorouracil, Oxaliplatin, Irinotecan -FOI), provides the best clinical benefit in pancreatic ductal adenocarcinoma (PDAC) patients. In this study we ...explore the role of miRNAs (MIR) as modulators of chemosensitivity to identify potential biomarkers of response. We find that 41 and 84 microRNA inhibitors enhance the sensitivity of Capan1 and MiaPaCa2 PDAC cells respectively. These include a MIR1307-inhibitor that we validate in further PDAC cell lines. Chemotherapy-induced apoptosis and DNA damage accumulation are higher in MIR1307 knock-out (MIR1307KO) versus control PDAC cells, while re-expression of MIR1307 in MIR1307KO cells rescues these effects. We identify binding of MIR1307 to CLIC5 mRNA through covalent ligation of endogenous Argonaute-bound RNAs cross-linking immunoprecipitation assay. We validate these findings in an in vivo model with MIR1307 disruption. In a pilot cohort of PDAC patients undergoing FOLFIRONX chemotherapy, circulating MIR1307 correlates with clinical outcome.
The transcriptional repressor BCL6 is an oncogenic driver found to be deregulated in lymphoid malignancies. Herein, we report the optimization of our previously reported benzimidazolone molecular ...glue-type degrader CCT369260 to CCT373566, a highly potent probe suitable for sustained depletion of BCL6 in vivo. We observed a sharp degradation SAR, where subtle structural changes conveyed the ability to induce degradation of BCL6. CCT373566 showed modest in vivo efficacy in a lymphoma xenograft mouse model following oral dosing.
Medulloblastoma (MB) is the most common malignant pediatric brain tumor. While the pathways that are deregulated in MB remain to be fully characterized, amplification and/or overexpression of the ...MYCN gene, which is has a critical role in cerebellar development as a regulator of neural progenitor cell fate, has been identified in several MB subgroups. Phenotypically, aberrant expression of MYCN is associated with the large-cell/anaplastic MB variant, which accounts for 5-15% of cases and is associated with aggressive disease and poor clinical outcome. To better understand the role of MYCN in MB in vitro and in vivo and to aid the development of MYCN-targeted therapeutics we established tumor-derived neurosphere cell lines from the GTML (Glt1-tTA/TRE-MYCN-Luc) genetically engineered mouse model. A fraction of GTML neurospheres were found to be growth factor independent, expressed CD133 (a marker of neural stem cells), failed to differentiate upon MYCN withdrawal and were highly tumorigenic when orthotopically implanted into the cerebellum. Principal component analyzes using single cell RNA assay data suggested that the clinical candidate aurora-A kinase inhibitor MLN8237 converts GTML neurospheres to resemble non-MYCN expressors. Correlating with this, MLN8237 significantly extended the survival of mice bearing GTML MB allografts. In summary, our results demonstrate that MYCN plays a critical role in expansion and survival of aggressive MB-propagating cells, and establish GTML neurospheres as an important resource for the development of novel therapeutic strategies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
MicroRNAs are small molecules which regulate gene expression post-transcriptionally and aberrant expression of several miRNAs is associated with neuroblastoma, a childhood cancer arising from ...precursor cells of the sympathetic nervous system. Amplification of the MYCN transcription factor characterizes the most clinically aggressive subtype of this disease, and although alteration of p53 signaling is not commonly found in primary tumors, deregulation of proteins involved in this pathway frequently arise in recurrent disease after pharmacological treatment. TH-MYCN is a well-characterized transgenic model of MYCN-driven neuroblastoma which recapitulates many clinicopathologic features of the human disease. Here, we evaluate the dysregulation of miRNAs in tumors from TH-MYCN mice that are either wild-type (TH-MYCN) or deficient (TH-MYCN/p53ER(TAM)) for the p53 tumor suppressor gene.
We analyzed the expression of 591 miRNAs in control (adrenal) and neuroblastoma tumor tissues derived from either TH-MYCN or TH-MYCN/p53ER(TAM) mice, respectively wild-type or deficient in p53. Comparing miRNA expression in tumor and control samples, we identified 159 differentially expressed miRNAs. Using data previously obtained from human neuroblastoma samples, we performed a comparison of miRNA expression between murine and human tumors to assess the concordance between murine and human expression data. Notably, the miR-17-5p-92 oncogenic polycistronic cluster, which is over-expressed in human MYCN amplified tumors, was over-expressed in mouse tumors. Moreover, analyzing miRNAs expression in a mouse model (TH-MYCN/p53ER(TAM)) possessing a transgenic p53 allele that drives the expression of an inactive protein, we identified miR-125b-3p and miR-676 as directly or indirectly regulated by the level of functional p53.
Our study represents the first miRNA profiling of an important mouse model of neuroblastoma. Similarities and differences in miRNAs expression between human and murine neuroblastoma were identified, providing important insight into the efficacy of this mouse model for assessing miRNA involvement in neuroblastoma and their potential effectiveness as therapeutic targets.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The early identification of children presenting ALK(F1174L)-mutated neuroblastoma, which are associated with resistance to the promising ALK inhibitor crizotinib and a marked poorer prognosis, has ...become a clinical priority. In comparing the radiology of the novel Th-ALK(F1174L)/Th-MYCN and the well-established Th-MYCN genetically-engineered murine models of neuroblastoma using MRI, we have identified a marked ALK(F1174L)-driven vascular phenotype. We demonstrate that quantitation of the transverse relaxation rate R2* (s(-1)) using intrinsic susceptibility-MRI under baseline conditions and during hyperoxia, can robustly discriminate this differential vascular phenotype, and identify MYCN-driven tumors harboring the ALK(F1174L) mutation with high specificity and selectivity. Intrinsic susceptibility-MRI could thus potentially provide a non-invasive and clinically-exploitable method to help identifying children with MYCN-driven neuroblastoma harboring the ALK(F1174L) mutation at the time of diagnosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Purpose of study: CHK1 and WEE1 are critical determinants of the G2/M cell cycle checkpoint. The G2/M checkpoint is crucial to enable DNA repair caused by endogenous and exogenous stress. We ...hypothesised that simultaneous inhibition of CHK1 and WEE1 would cause cell death in TP53 mutated cell lines because of the associated replication stress due to an abrogated G1/S checkpoint.
Experimental procedures: The potentiation index of the combination of SRA737 and adavosertib was calculated in six cancer cell lines harbouring TP53 mutations (OVCAR3, COV505, BT20, MDA-MB-436 and CAPAN1, MIAPaCa2) using Cell Titre Blue assays. Cell death and DNA damage was quantified using c-PARP, p-CHK1 Ser345 and gamma H2AX using western blot analysis. In-vivo studies evaluated the efficacy of vehicle control, SRA737, adavosertib and the combination in OVCAR3 and MDA-MB-436 xenograft models. We further explored the mechanisms of DNA damage repair caused by the single agents and the combination using two functional fluorescent multiplex DNA repair assay platforms in OVCAR3 and MDA-MB-436 cells; Exy-SPOT (base excision repair/nucleotide excision repair) and Next-SPOT (double strand break repair).
Results: The potentiation index of the addition of SRA737 to adavosertib were 19, 8.6, 8.8, 9.7, 27.2 and 5.7 for COV504, OVCAR3, BT20, MDA-MB-436, CAPAN1 and MIAPaCa2 respectively. The combination of both drugs at single agent GI50 concentrations caused increase in c-PARP, p-CHK1 Ser345 and gamma H2AX in all cell lines studied. We chose to explore the combination in the TP53 mutated OVCAR3 and the TP53 and BRCA1 mutated MDA-MB-436 cell lines. In the OVCAR3 xenograft model, the combination of SRA737 and adavosertib caused tumour regressions and tumour volumes were significantly smaller than controls, p<0001. The combination caused a lesser but significant degree of reduction in the tumour volume in the MDA-MB-436 xenograft model compared to control p=0.003. There was less than a 20% loss of mouse body weight in treatment arms of both xenograft models. Functional DNA damage repair analysis showed the combination of SRA737 and adavosertib caused a significantly greater reduction in oxidative stress related base excision repair (Etheno and 8oxoG lesions) as well as double stranded break repair via alternative end joining in the OVCAR3 cell lines compared to MDA-MB-436 cells, p=0.009, p=0.014 and p=0.005 respectively.
Conclusions: The combination of SRA737 and adavosertib caused cell death in TP53 mutated cell lines. There were differences in the DNA damage repair mechanisms in across cell lines and the combination showed significant activity in the xenograft models studied. The combination of SRA737 and adavosertib warrants further evaluation for the treatment of selected TP53 mutated cancers.
Citation Format: Adam Stewart, Lisa Pickard, Albert E. Hallsworth, Sylvie Sauvaigo, Giovanna Muggiolu, Florence Raynaud, UDAI BANERJI. A study of combinatorial growth inhibition, cell death and DNA damage repair caused by CHK1 inhibitor SRA737 and WEE1 inhibitor adavosertib in TP53 mutated cell lines abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1010.
The ALKF1174L mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ...ALKF1174L in the neural crest. Compared to ALKF1174L and MYCN alone, co-expression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance, and enhanced lethality. ALKF1174L/MYCN tumors exhibited increased MYCN dosage due to ALKF1174L-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2 overcame the resistance of ALKF1174L/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALKF1174L in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.
► A murine model of ALKF1174L/MYCN neuroblastoma is presented ► ALK potentiates MYCN-driven oncogenesis at multiple levels in neuroblastoma ► ALKF1174L/MYCN tumors exhibit therapy resistance in vivo ► mTOR inhibition circumvents crizotinib resistance in vivo
In this age of molecularly targeted drug discovery, robust techniques are required to measure pharmacodynamic (PD) responses in tumors so that drug exposures can be associated with their effects on ...molecular biomarkers and efficacy. Our aim was to develop a rapid screen to monitor PD responses within xenografted human tumors as an important step towards a clinically applicable technology. Currently there are various methods available to measure PD end points, including immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction, gene expression profiling, and western blotting. These may require relatively large samples of tumor, surrogate tissue, or peripheral blood lymphocytes with subsequent analyses taking several days. The phosphoinositide 3-kinase (PI3-kinase) pathway is frequently deregulated in cancer and is also important in diabetes and autoimmune conditions. In this paper, optimization of the Meso Scale Discovery (MSD) (Gaithersburg, MD) platform to quantify changes in phospho-AKT and phospho-glycogen synthase kinase-3beta in response to a PI3-kinase inhibitor, LY294002, is described, initially in vitro and then within xenografted solid tumors. This method is highly practical with high throughput since large number of samples can be run simultaneously in 96-well format. The assays are robust (coefficient of variation for phospho-AKT 13.4%) and offer significant advantages (in terms of speed and quantitation) over western blots. This optimized procedure can be used for both in vitro and in vivo analysis, unlike an established fixed-cell ELISA with a time-resolved fluorescent end point.
The ALK(F1174L) mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ...ALK(F1174L) in the neural crest. Compared to ALK(F1174L) and MYCN alone, co-expression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance, and enhanced lethality. ALK(F1174L)/MYCN tumors exhibited increased MYCN dosage due to ALK(F1174L)-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2 overcame the resistance of ALK(F1174L)/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALK(F1174L) in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.
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