ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently has no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A mutated ovarian cancer cells. ARID1A mutational status correlates with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct ARID1A/EZH2 target, which is upregulated by EZH2 inhibition and contributes to the observed synthetic lethality by inhibiting PI3K/AKT signaling. Significantly, EZH2 inhibition causes regression of ARID1A mutated ovarian tumors in vivo. Together, these data demonstrate for the first time a synthetic lethality between ARID1A mutation and EZH2 inhibition. They indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for ARID1A mutated cancers.

• Enasidenib inhibits mIDH2, leading to leukemic cell differentiation with emergence of functional mIDH2 neutrophils in rrAML patients.• RAS pathway mutations and increased mutational burden overall are associated with a decreased response rate to mIDH2 inhibition.Recurrent mutations at R140 and R172 in isocitrate dehydrogenase 2 (IDH2) occur in many cancers, including ∼12% of acute myeloid leukemia (AML). In preclinical models these mutations cause accumulation of the oncogenic metabolite R-2-hydroxyglutarate (2-HG) and induce hematopoietic differentiation block. Single-agent enasidenib (AG-221/CC-90007), a selective mutant IDH2 (mIDH2) inhibitor, produced an overall response rate of 40.3% in relapsed/refractory AML (rrAML) patients with mIDH2 in a phase 1 trial. However, its mechanism of action and biomarkers associated with response remain unclear. Here, we measured 2-HG, mIDH2 allele burden, and co-occurring somatic mutations in sequential patient samples from the clinical trial and correlated these with clinical response. Furthermore, we used flow cytometry to assess inhibition of mIDH2 on hematopoietic differentiation. We observed potent 2-HG suppression in both R140 and R172 mIDH2 AML subtypes, with different kinetics, which preceded clinical response. Suppression of 2-HG alone did not predict response, because most nonresponding patients also exhibited 2-HG suppression. Complete remission (CR) with persistence of mIDH2 and normalization of hematopoietic stem and progenitor compartments with emergence of functional mIDH2 neutrophils were observed. In a subset of CR patients, mIDH2 allele burden was reduced and remained undetectable with response. Co-occurring mutations in NRAS and other MAPK pathway effectors were enriched in nonresponding patients, consistent with RAS signaling contributing to primary therapeutic resistance. Together, these data support differentiation as the main mechanism of enasidenib efficacy in relapsed/refractory AML patients and provide insight into resistance mechanisms to inform future mechanism-based combination treatment studies. (Blood. 2017;130(6):732-741)

Stromagenesis is a host reaction of connective tissue that, when induced in cancer, produces a progressive and permissive mesenchymal microenvironment, thereby supporting tumor progression. The stromal microenvironment is complex and comprises several cell types, including fibroblasts, the primary producers of the noncellular scaffolds known as extracellular matrices. The events that support tumor progression during stromagenesis are for the most part unknown due to the lack of suitable, physiologically relevant, experimental model systems. In this report, we introduce a novel in vivo-like three-dimensional system derived from tumor-associated fibroblasts at diverse stages of tumor development that mimic the stromagenic features of fibroblasts and their matrices observed in vivo. Harvested primary stromal fibroblasts, obtained from different stages of tumor development, did not retain in vivo stromagenic characteristics when cultured on traditional two-dimensional substrates. However, they were capable of effectively maintaining the tumor-associated stromal characteristics within three-dimensional cultures. In this study, we demonstrate that in vivo-like three-dimensional matrices appear to have the necessary topographical and molecular information sufficient to induce desmoplastic stroma differentiation of normal fibroblasts.

Fibroblasts secrete and organize extracellular matrix (ECM), which provides structural support for their adhesion, migration, and tissue organization, besides regulating cellular functions such as growth and survival. Cell-to-matrix interactions are vital for vertebrate development. Disorders in these processes have been associated with fibrosis, developmental malformations, cancer, and other diseases. This unit describes a method for preparing a three-dimensional matrix derived from fibroblastic cells; the matrix is three-dimensional, cell and debris free, and attached to a two-dimensional culture surface. Cell adhesion and spreading are normal on these matrices. This matrix can also be compressed into a two-dimensional matrix and solubilized to study the matrix biochemically. Culturing fibroblasts on traditional two-dimensional (2-D) substrates induces an artificial polarity between lower and upper surfaces of these normally nonpolar cells. Not surprisingly, fibroblast morphology and migration differ once suspended in three-dimensional (3-D) collagen gels (Friedl and Brocker, 2000). However, the molecular composition of collagen gels does not mimic the natural fibroblast (i.e., mesenchymal) microenvironment. Fibroblasts secrete and organize ECM, which provides structural support for their adhesion, migration, and tissue organization, in addition to regulating cellular functions such as growth and survival (Buck and Horwitz, 1987; Hay, 1991; Hynes, 1999; Geiger et al., 2001). Cell-to-matrix interactions are vital for vertebrate development. Disorders in these processes have been associated with fibrosis, developmental malformations, cancer (i.e., desmoplastic tumor microenvironment), and other diseases (Rybinski et al., 2014). This unit describes methods for generating tissue culture surfaces coated with a fibroblast-derived 3-D ECM produced and deposited by both established and primary fibroblasts. The matrices closely resemble in vivo mesenchymal matrices and are composed mainly of fibronectin fibrillar lattices. Utilizing in vivo-like 3-D matrices as substrates allows the acquisition of information that is physiologically relevant to cell-matrix interactions, structure, function, and signaling, which differ from data obtained by culturing cells on conventional 2-D substrates in vitro (Cukierman et al., 2001). These protocols were initially derived from methods described in UNIT 10.4, which were modified to obtain fibroblast-derived 3-D matrices and to characterize cellular responses to them. The basic approach is to allow fibroblasts to produce their own 3-D matrix (see Basic Protocol). For this purpose, fibroblasts are plated and maintained in culture in a confluent state. After 5 to 9 days, unextracted 3-D matrix cultures can be sorted into normal or activated (i.e., myofibroblastic, fibrotic or desmoplastic) phenotypes (see Support Protocol 1) or matrices are denuded of cells, and cellular remnants are removed. Such extraction results in an intact fibroblast-derived 3-D matrix that is free of cellular deb...

Mutations in the gene encoding isocitrate dehydrogenase 2 (IDH2) occur in several types of cancer, including acute myeloid leukemia (AML). In model systems, mutant IDH2 causes hematopoietic differentiation arrest. Enasidenib, a selective small-molecule inhibitor of mutant IDH2, produces a clinical response in 40% of treated patients with relapsed/refractory AML by promoting leukemic cell differentiation. Here, we studied the clonal basis of response and acquired resistance to enasidenib treatment. Using sequential patient samples, we determined the clonal structure of hematopoietic cell populations at different stages of differentiation. Before therapy, IDH2-mutant clones showed variable differentiation arrest. Enasidenib treatment promoted hematopoietic differentiation from either terminal or ancestral mutant clones; less frequently, treatment promoted differentiation of nonmutant cells. Analysis of paired diagnosis/relapse samples did not identify second-site mutations in IDH2 at relapse. Instead, relapse arose by clonal evolution or selection of terminal or ancestral clones, thus highlighting multiple bypass pathways that could potentially be targeted to restore differentiation arrest. These results show how mapping of clonal structure in cell populations at different stages of differentiation can reveal the response and evolution of clones during treatment response and relapse.