Tim Failes

Comparative drug screening in NUT midline carcinoma

Background:The NUT midline carcinoma (NMC) is a rare but fatal cancer for which systematic testing of therapy options has never been performed.Methods:On the basis of disease biology, we compared the efficacy of the CDK9 inhibitor flavopiridol (FP) with a panel of anticancer agents in NMC cell lines and mouse xenografts.Results:In vitro anthracyclines, topoisomerase inhibitors, and microtubule poisons were among the most cytotoxic drug classes for NMC cells, while efficacy of the bromodomain inhibitor JQ1 varied considerably between lines carrying different BRD4 (bromodomain-containing protein 4)–NUT (nuclear protein in testis) translocations. Efficacy of FP was comparable to vincristine and doxorubicin, drugs that have been previously used in NMC patients. All three compounds showed significantly better activity than etoposide and vorinostat, agents that have also been used in NMC patients. Statins and antimetabolites demonstrated intermediate single-agent efficacy. In vivo, vincristine significantly inhibited tumour growth in two different NMC xenografts. Flavopiridol in vivo was significantly effective in one of the two NMC xenograft lines, demonstrating the biological heterogeneity of this disease.Conclusions:These results demonstrate that FP may be of benefit to a subset of patients with NMC, and warrant a continued emphasis on microtubule inhibitors, anthracyclines, and topoisomerase inhibitors as effective drug classes in this disease.

High-Throughput Screening of Human Leukemia Xenografts to Identify Dexamethasone Sensitizers:

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Glucocorticoids (e.g., dexamethasone) form a critical component of chemotherapy regimens for pediatric ALL, and the initial response to glucocorticoid therapy is a major prognostic factor, where resistance is predictive of poor outcome. We have previously established a clinically relevant ALL xenograft model, consisting of primary pediatric ALL biopsies engrafted into immune-deficient mice, in which in vitro and in vivo dexamethasone sensitivity significantly correlated with patient outcome. In this study, we used high-throughput screening (HTS) to identify novel compounds that reverse dexamethasone resistance in a xenograft (ALL-19) derived from a chemoresistant pediatric ALL patient that is representative of the most common pediatric ALL subtype (B-cell precursor [BCP-ALL]). The compound 2-(4-chlorophenoxy)-2-methyl-N-(2-(piperidin-1-yl)phenyl)propanamide showed little cytotoxic activity alone (IC50 = 31 µM), but when combined with dexamethasone, it caused a marked decrease in cell viability. Fixed-ratio combination assays were performed against a broad panel of dexamethasone-resistant and -sensitive xenografts representative of BCP-ALL, T-cell ALL, and Mixed Lineage Leukemia–rearranged ALL, and synergy was observed in six of seven xenografts. We describe here the development of a novel 384-well cell-based high-throughput screening assay for identifying potential dexamethasone sensitizers using a clinically relevant ALL xenograft model.

Integration of genomics, high throughput drug screening, and personalized xenograft models as a novel precision medicine paradigm for high risk pediatric cancer

ABSTRACT Pediatric high grade gliomas (HGG) are primary brain malignancies that result in significant morbidity and mortality. One of the challenges in their treatment is inter- and intra-tumoral heterogeneity. Precision medicine approaches have the potential to enhance diagnostic, prognostic and/or therapeutic information. In this case study we describe the molecular characterization of a pediatric HGG and the use of an integrated approach based on genomic, in vitro and in vivo testing to identify actionable targets and treatment options. Molecular analysis based on WGS performed on initial and recurrent tumor biopsies revealed mutations in TP53, TSC1 and CIC genes, focal amplification of MYCN, and copy number gains in SMO and c-MET. Transcriptomic analysis identified increased expression of MYCN, and genes involved in sonic hedgehog signaling proteins (SHH, SMO, GLI1, GLI2) and receptor tyrosine kinase pathways (PLK, AURKA, c-MET). HTS revealed no cytotoxic efficacy of SHH pathway inhibitors while sensitivity was observed to the mTOR inhibitor temsirolimus, the ALK inhibitor ceritinib, and the PLK1 inhibitor BI2536. Based on the integrated approach, temsirolimus, ceritinib, BI2536 and standard therapy temozolomide were selected for further in vivo evaluation. Using the PDX animal model (median survival 28 days) we showed significant in vivo activity for mTOR inhibition by temsirolimus and BI2536 (median survival 109 and 115.5 days respectively) while ceritinib and temozolomide had only a moderate effect (43 and 75.5 days median survival respectively). This case study demonstrates that an integrated approach based on genomic, in vitro and in vivo drug efficacy testing in a PDX model may be useful to guide the management of high risk pediatric brain tumor in a clinically meaningful timeframe.

Abstract B23: Selective and rapid killing of mixed lineage leukemia and CALM-AF10 leukemia by SM7, a novel small molecule identified by chemical library screening

Translocations involving the Mixed Lineage Leukemia (MLL) gene define the major proportion of infant leukemia, a disease that is characterised by a dismal prognosis due to treatment toxicity and therapy resistance. There is an urgent need for the development of less toxic, more selective and targeted therapies for this disease. In this study, we performed a cell-based chemical library screen on an infant MLL cell line, PER-485, in order to identify selective inhibitors of MLL leukemia. After screening initial hits for viability against a panel of 25 cell lines including MLL rearranged and wild-type MLL leukemia, solid tumors and control cells, SM7 was identified as a compound that significantly and selectively decreased the viability of MLL-rearranged (four out of seven) and related CALM-AF10 (two out of two) leukemia cell lines, without killing cell lines derived from wild-type MLL leukemia, solid tumours or normal cells. In contrast to currently developed MLL-inhibitors, the effect of SM7 in suppressing the characteristic MLL-driven gene expression signature in MLL-rearranged cells, with downregulation of leukemogenic HoxA9 and Meis1 expression, occurred within an hour after treatment. The transcript levels of cMyc and Bcl2, important survival proteins regulated by HoxA9, were also rapidly decreased in response to SM7. SM7 induced a rapid caspase-dependent apoptosis, with mitochondrial depolarization within twenty four hours of treatment. This is distinct from the action of other previously described MLL small molecule inhibitors. The rapid and selective toxicity of SM7 towards MLL-rearranged and CALM-AF10 leukemia cell lines suggests an important new avenue in the development of targeted therapies for this disease. Citation Format: Daria Chudakova, Klaartje Somers, Shiloh Middlemiss, Victoria Wen, Alan Kwek, Bing Liu, Molly Clifton, Sukey Pan, Sarah Cruikshank, Marissa Scandlyn, Tim Failes, Wendy Hoang, Ursula Kees, Olga Chernova, Andrei V. Gudkov, Michelle Haber, Murray Norris, Michelle Henderson. Selective and rapid killing of mixed lineage leukemia and CALM-AF10 leukemia by SM7, a novel small molecule identified by chemical library screening. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B23.

Abstract 3660: Discovery of a small molecule TUBB3/βIII-tubulin modulator in lung cancer

Background: Non-small Cell Lung Cancer (NSCLC) survival rates are dismal and chemotherapy resistance is a significant clinical problem. βIII-tubulin (encoded by TUBB3 gene) is aberrantly expressed and is associated with chemoresistance and tumor aggressiveness in NSCLC (1), where it has been identified as a bona fide target for chemosensitisation (2,3). In order to understand how TUBB3/βIII-tubulin is regulated in NSCLC cells we sought to identify chemical small molecules that can modulate its expression. Methods: H460 cells expressing a TUBB3 or GAPDH promoter-luciferase reporter construct were generated and used in drug screening and promoter activity testing. To isolate modulators of TUBB3 promoter activity, a cell-based screen of libraries of diverse chemical small molecules (30K) and FDA-approved bioactives (3680) was performed. Cell viability, growth and proliferation were measured using standard methods. Cell cycle was assessed using flow cytometry, and gene and protein expression by RT-PCR and Western blotting, respectively. Microtubule morphology was assessed using immunostaining and confocal microscopy. Drug-treated clonogenic assays were used to quantitate changes in drug sensitivity. Results: Based upon their ability to modulate TUBB3 promoter activity, we identified two hit compounds, CCI01 and CCI02, as well as the bioactive compound, RITA. For all three leads we observed: 1) repression in TUBB3 promoter activity which was not a result of cell cytotoxicity; 2) no effects on cell cycle or viability, but instead cytostatic effects; 3) significantly enhanced TUBB3 expression in a time and dose-dependent manner. TUBB3 gene enhancement was translated at the protein level in CCI01 treated H460, but not in CCI02 or RITA treated cells. Additionally, CCI01 did not alter microtubule morphology but enhanced βIII-tubulin immunostaining in two independent NSCLC cell lines, H460 and H1299, compared to control. Importantly, CCI01 enhanced βIII-tubulin expression was functional and led to a significant decrease in in vitro sensitivity to DNA-damaging and tubulin-binding agents in H460 cells. Conclusion: A novel lead small molecule TUBB3/βIII-tubulin enhancer has been identified that is able to increase expression of βIII-tubulin in NSCLC and significantly reduce sensitivity to tubulin binding and DNA-damaging agents. Implications: Identification of a modulator of TUBB3/βIII-tubulin expression will provide a valuable research tool to probe βIII-tubulin regulation. (1) Kavallaris. Nature Rev Cancer, 10:194-204, 2010 (2) McCarroll et al., Cancer Res 70:4995-5003, 2010 (3) Gan et al., Cancer Res. 67:9356-9363, 2007 Citation Format: Felicity Chao Lin Kao, Tim Failes, Greg M. Arndt, Murray Norris, Maria Kavallaris. Discovery of a small molecule TUBB3/βIII-tubulin modulator in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3660. doi:10.1158/1538-7445.AM2015-3660

Abstract 4550: High throughput screen to identify compounds that reverse glucocorticoid resistance in pediatric leukemia.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Although cure rates are approaching 85%, it remains one of the most common causes of death from disease in children. Glucocorticoids (e.g. dexamethasone) form a critical component of chemotherapy regimens for pediatric ALL and the initial response to glucocorticoid therapy is a major prognostic factor, where resistance is predictive of poor outcome. We have previously established a clinically relevant ALL xenograft model, consisting of primary pediatric ALL biopsies engrafted in immune-deficient mice, in which in vitro and in vivo dexamethasone sensitivity significantly correlate with patient outcome. In this study we used a high throughput screen (HTS) to identify novel small compounds that reverse glucocorticoid resistance. A xenograft (ALL-19) derived from an aggressive and chemoresistant pediatric ALL that induced early fatality in the patient, and is representative of the most common pediatric ALL subtype (B-cell precursor, BCP-ALL, or c-ALL) was used to identify compounds that potentially will have wide applicability. Cells were exposed in vitro to dexamethasone at a concentration ineffective in cell killing against this xenograft (1 μM), but which is around 100-fold greater than the IC50 for other (sensitive) xenografts, simultaneously with one of 40,000 HTS library compounds (10 μM). Effective compounds were then tested alone and in the presence of dexamethasone, to identify those that sensitized the xenograft to glucocorticoids, rather than those that were toxic alone. The compound 2-(4-chlorophenoxy)-2-methyl-N-(2-(piperidin-1-yl)phenyl)propanamide showed little activity alone (IC50 22 μM), but when combined with dexamethasone caused a marked decrease in cell viability. Pre-incubating ALL-19 xenograft cells with either the compound or dexamethasone did not increase the sensitizing effect. Fixed-ratio combination assays were performed against a broad panel of dexamethasone-resistant and -sensitive xenografts representative of BCP-ALL, T-ALL and MLL-rearranged ALL, in order to determine whether the compound was broadly active. CalcuSyn software was employed to determine combination indices, and synergy was observed in all 4 BCP-ALL xenografts tested, which included a dexamethasone-sensitive BCP-ALL and a dexamethasone-resistant Philadelphia chromosome-positive ALL. Synergy was also observed in dexamethasone-resistant MLL-rearranged ALL xenograft cells. Additionally, despite an antagonistic effect observed in a dexamethasone-resistant T-ALL xenograft, the compound potentiated the effects of dexamethasone in a glucocorticoid-sensitive T-ALL. In conclusion, using a clinically relevant xenograft model and a HTS, this study has identified a novel compound that reverses dexamethasone resistance in BCP-ALL xenografts, and may have applications in other leukemia subtypes. Citation Format: Cara Toscan, Tim Failes, Greg Arndt, Richard Lock. High throughput screen to identify compounds that reverse glucocorticoid resistance in pediatric leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4550. doi:10.1158/1538-7445.AM2013-4550