Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane,Thomas Brunner, Ella Bossy-Wetzel and Douglas R. GreenThis protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector etal.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. Thisthree-volume set is now out of print; however, some of the microscopy methods wererepublished in Basic Methods in Microscopy, by David L. Spector and Robert D.Goldman.
We have identified 5-(3-chlorothiophen-2-yl)-3-(4-trifluoromethylphenyl)-1,2,4-oxadiazole (1d) as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 1d has good activity against several breast and colorectal cancer cell lines but is inactive against several other cancer cell lines. In a flow cytometry assay, treatment of T47D cells with 1d resulted in arrest of cells in the G(1) phase, followed by induction of apoptosis. SAR studies of 1d showed that the 3-phenyl group can be replaced by a pyridyl group, and a substituted five-member ring in the 5-position is important for activity. 5-(3-Chlorothiophen-2-yl)-3-(5-chloropyridin-2-yl)-1,2,4-oxadiazole (4l) has been found to have in vivo activity in a MX-1 tumor model. Using a photoaffinity agent, the molecular target has been identified as TIP47, an IGF II receptor binding protein. Therefore, our cell-based chemical genetics approach for the discovery of apoptosis inducers can identify potential anticancer agents as well as their molecular targets.
Abstract Extrachromosomal DNA (ecDNA) presents a major challenge for precision medicine, contributing to poor survival for patients with oncogene-amplified tumours. EcDNA renders tumours resistant to targeted treatments by facilitating massive transcription of oncogenes and rapid genome evolution. At present, there are no ecDNA- specific treatments. Here we show that enhancing transcription replication conflict enables targeted elimination of ecDNA-containing cancers, exposing an actionable vulnerability. Stepwise analyses of ecDNA transcription reveal landscapes of pervasive RNA transcription and associated single-stranded DNA, leading to excessive transcription replication conflicts and replication stress (RS) compared to chromosomal loci. Nucleotide incorporation onto growing DNA strands is markedly slower on ecDNA, and RS is significantly higher in ecDNA-containing tumours regardless of cancer type or oncogene cargo. Replication Protein A2 phosphorylated on serine 33, a mediator of DNA damage repair that binds single-stranded DNA, shows elevated localization on ecDNA in a transcription dependent manner, along with increased DNA double strand breaks, and activation of the S-phase checkpoint kinase, CHK1. Genetic or pharmacological CHK1 inhibition abrogates the DNA replication check point, causing extensive and preferential tumour cell death in ecDNA-containing tumours as they enter S-phase. To exploit this vulnerability, we develop a highly selective, potent, and bioavailable oral CHK1 inhibitor, BBI-2779, and demonstrate that it preferentially kills ecDNA-containing tumour cells. In a gastric cancer model containing FGFR2 on ecDNA, BBI-2779, suppresses tumour growth and prevents ecDNA-mediated acquired resistance to the pan-FGFR inhibitor infigratinib, resulting in potent and sustained tumour regression in mice. These results reveal transcription-replication conflict as an ecDNA-generated vulnerability that can be targeted as an ecDNA-directed therapy and suggest that synthetic lethality of excess can be exploited as a strategy for treating cancer.
Checkpoint inhibition has transformed immunotherapy by alleviating T cell exhaustion in a subset of patients. However, an important component of effective immune targeting to expand the benefit of immune response requires engagement of both innate and adaptive responses. Our understanding of safe and effective engagement of the innate immune system is evolving, with multiple preclinical and clinical agents targeting pathways such the Toll-like Receptors. Here we disclose the structure and preclinical activity of LHC165, a benzonapthyridine TLR7 agonist that is adsorbed to aluminum hydroxide. The interaction between LHC165 and aluminum hydroxide allows for a slow release from the injection site resulting in improved efficacy in mouse models compared with free LHC165. This localization allows for immune activation at the site of the tumor and also results in lower systemic exposure and cytokine induction. Intratumoral studies in syngeneic preclinical studies show single agent activity and a benefit when dosed in combination with checkpoint blockade. LHC165 as a single agent and in combination with PDR001 is currently enrolling patients with advanced malignancies in CLHC165X2101.Citation Format: Jonathan A. Deane, German A. Cortez, Chun Li, Nora Eifler, Shailaja Kasibhatla, Nehal Parikh, Shifeng Pan, Steven Bender. Identification and characterization of LHC165, a TLR7 agonist designed for localized intratumoral therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4128.
// Jinyun Chen 1 , Franklin Chung 1 , Guizhi Yang 1 , Minying Pu 1 , Hui Gao 1 , Wei Jiang 2 , Hong Yin 2 , Vladimir Capka 2 , Shailaja Kasibhatla 3 , Bryan Laffitte 3 , Savina Jaeger 1 , Raymond Pagliarini 1 , Yaoyu Chen 1 and Wenlai Zhou 1 1 Oncology, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, United States 2 Analytic Science, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, United States 3 The Genomics Institute of the Novartis Research Foundation, San Diego, California, United States Correspondence: Yaoyu Chen, email : yaoyu.chen@novartis.com, email: // Wenlai Zhou, email: // Keywords : PHGDH, breast cancer cells, in vivo Received : October 24, 2013 Accepted : November 24, 2013 Published : November 26, 2013 Abstract Cancer cells rely on aerobic glycolysis to maintain cell growth and proliferation via the Warburg effect. Phosphoglycerate dehydrogenase (PHDGH) catalyzes the first step of the serine biosynthetic pathway downstream of glycolysis, which is a metabolic gatekeeper both for macromolecular biosynthesis and serine-dependent DNA synthesis. Here, we report that PHDGH is overexpressed in many ER-negative human breast cancer cell lines. PHGDH knockdown in these cells leads to a reduction of serine synthesis and impairment of cancer cell proliferation. However, PHGDH knockdown does not affect tumor maintenance and growth in established breast cancer xenograft models, suggesting that PHGDH-dependent cancer cell growth may be context-dependent. Our findings suggest that other mechanisms or pathways may bypass exclusive dependence on PHGDH in established human breast cancer xenografts, indicating that PHGDH is dispensable for the growth and maintenance and of tumors in vivo .
The selective inhibition of RET kinase as a treatment for relevant cancer types including lung adenocarcinoma has garnered considerable interest in recent years and prompted a variety of efforts toward the discovery of small-molecule therapeutics. Hits uncovered via the analysis of archival kinase data ultimately led to the identification of a promising pyrrolo[2,3-d]pyrimidine scaffold. The optimization of this pyrrolo[2,3-d]pyrimidine core resulted in compound 1, which demonstrated potent in vitro RET kinase inhibition and robust in vivo efficacy in RET-driven tumor xenografts upon multiday dosing in mice. The administration of 1 was well-tolerated at established efficacious doses (10 and 30 mg/kg, po, qd), and plasma exposure levels indicated a minimal risk of KDR or hERG inhibition in vivo, as evaluated by Miles assay and free plasma concentrations, respectively.
Abstract Non-small cell lung cancers (NSCLC) harboring anaplastic lymphoma kinase (ALK) gene rearrangements are sensitive to the ALK tyrosine kinase inhibitor (TKI) crizotinib. However, these cancers invariably relapse due to the development of resistance, and approximately 1/3 of such cancers develop resistance mutations within the ALK tyrosine kinase domain. Here we report the preclinical evaluation of the next-generation ALK TKI, LDK378 in the setting of crizotinib resistance. Using EML4-ALK mutant Ba/F3 cellular models, in vivo models of acquired resistance to crizotinib, and novel cell lines established from biopsies of crizotinib-resistant NSCLC patients, we have examined the efficacy of LDK378 in crizotinib-naïve and -resistant ALK-positive cancers. These studies reveal that LDK378 is more potent than crizotinib and effectively overcomes resistance in vitro and in vivo. In particular, LDK378 inhibits ALK harboring crizotinib resistance mutations, including L1196M, G1269A, I1171T and S1206Y. Cell lines derived from crizotinib-resistant biopsies were sensitive to LDK378, including one that did not harbor an ALK resistance mutation and was also sensitive to crizotinib, suggesting that some crizotinib-resistant cancers with wildtype ALK are still sensitive to complete ALK inhibition. We observed that LDK378 did not effectively overcome two crizotinib-resistant ALK mutations, G1202R and F1174C ALK, and mutations in one of these residues was identified in 5 out of 11 biopsies from patients with acquired resistance to LDK378. Altogether our results demonstrate that LDK378 can overcome many mechanisms of crizotinib resistance, consistent with emerging clinical data showing marked efficacy of LDK378 in patients with crizotinib-resistant disease. Citation Format: Luc Friboulet, Nanxin Li, Ryohei Katayama, Christian C. Lee, Justin F. Gainor, Adam S. Crystal, Pierre-Yves Michellys, Mark M. Awad, Noriko Yanagitani, Sungjoon Kim, AnneMarie Pferdekamper, Jie Li, Shailaja Kasibhatla, Frank Sun, Xiuying Sun, Su Hua, Peter McNamara, Sidra Mahmood, Elizabeth L. Lockerman, Naoya Fujita, Makoto Nishio, Jennifer L. Harris, Alice T. Shaw, Jeffrey A. Engelman. The ALK inhibitor LDK378 overcomes crizotinib resistance in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 957. doi:10.1158/1538-7445.AM2014-957
