Establishment of Patient-Derived Succinate Dehydrogenase-Deficient Gastrointestinal Stromal Tumor Models for Predicting Therapeutic Response

Abstract

Purpose: Gastrointestinal stromal tumor (GIST) is the most common sarcoma of the gastrointestinal tract, with mutant succinate dehydrogenase (SDH) subunits (A-D) comprising less than 7.5% (i.e., 150-200/year) of new cases annually in the United States. Contrary to GISTs harboring KIT or PDGFRA mutations, SDH-mutant GISTs affect adolescents/young adults, often metastasize, and are frequently resistant to tyrosine kinase inhibitors (TKI). Lack of human models for any SDH-mutant tumors, including GIST, has limited molecular characterization and drug discovery.

Experimental design: We describe methods for establishing novel patient-derived SDH-mutant (mSDH) GIST models and interrogated the efficacy of temozolomide on these tumor models in vitro and in clinical trials of patients with mSDH GIST.

Results: Molecular and metabolic characterization of our patient-derived mSDH GIST models revealed that these models recapitulate the transcriptional and metabolic hallmarks of parent tumors and SDH deficiency. We further demonstrate that temozolomide elicits DNA damage and apoptosis in our mSDH GIST models. Translating our in vitro discovery to the clinic, a cohort of patients with SDH-mutant GIST treated with temozolomide (n = 5) demonstrated a 40% objective response rate and 100% disease control rate, suggesting that temozolomide represents a promising therapy for this subset of GIST.

Conclusions: We report the first methods to establish patient-derived mSDH tumor models, which can be readily employed for understanding patient-specific tumor biology and treatment strategies. We also demonstrate that temozolomide is effective in patients with mSDH GIST who are refractory to existing chemotherapeutic drugs (namely, TKIs) in clinic for GISTs, bringing a promising treatment option for these patients to clinic.See related commentary by Blakely et al., p. 3.

Figure 1.. Establishment and characterization of SDHA, SDHB, and SDHC-mutant human GIST models.

A. Schematic representation of workflow of establishment of patient-derived mSDH GIST models and validation for recapitulation of essential features of parent tumors. B. Micrographs of mSDH GIST models SD-437A, SD-424B, and SD-435C propagated in adherent conditions on a laminin-rich HTB9 matrix (2D) or in non-adherent conditions for 7 days as spheroids on Poly-HEMA coated wells (3D). Scale bar, 50 μm. C. Immunofluorescence staining of KIT (red) and DOG-1 (green) in parent tumors and mSDH GIST models grown in adherent and in non-adherent conditions. D. Immunoblot analysis confirming expression of SDHA and SDHB protein in KIT-mutant/SDH-WT (wildtype) GIST882 cells and in mSDH GIST models. α-tubulin was used as a loading control. E. Immunofluorescence staining of SDHB (green) and DAPI (blue) in KIT-mutant/SDH-WT (wildtype) GIST882 cells and in mSDH GIST models. Scale bar, 50 μm.

Figure 2.. Metabolic profiles of mSDH GIST models recapitulate succinate dehydrogenase deficiency.

A. Per cell abundance of succinate relative to GIST882 in mSDH GIST models grown for 48 h (n = 3). One-way ANOVA (multiple comparisons) was performed for statistical analysis with ****, P<0.0001. B. Ratio of intracellular succinate-to-fumarate concentrations relative to GIST882 in mSDH GIST models grown for 48 h (n = 3). One-way ANOVA (multiple comparisons) was performed for statistical analysis with P>0.05 (ns), ****, P<0.0001. C. Oxygen consumption rate (OCR) trace of intact mSDH GIST models (n = 5). D. Basal respiration rate of intact mSDH GIST models (n = 5). One-way ANOVA (multiple comparisons) was performed for statistical analysis with ****, P<0.0001. E. Maximal uncoupled respiration driven by succinate relative to WT in permeabilized mSDH GIST models (n = 5). One-way ANOVA was performed. ***, P<0.001; ****, P<0.0001. Maximal uncoupled respiration driven by NADH relative to WT in permeabilized mSDH GIST models (n = 5). One-way ANOVA was performed. ***, P<0.001; ****, P<0.0001. F. Ratio of intracellular α-ketoglutarate (α-KG) to citrate relative to GIST882 in mSDH GIST models grown for 48 h (n = 3). G. Atom transition diagram of reductive glutamine catabolism using a [U-¹³C₅] glutamine tracer. Open circles represent ¹²C, closed circles represent ¹³C carbon atoms. H. Percent labeling of M5 citrate from [U-¹³C₅]-glutamine in mSDH GIST models grown for 48 h (n = 3). Data are represented as mean ± SEM with biological replicates as indicated. One-way ANOVA was performed for statistical analysis with P>0.05 (ns), *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Figure 3.. Transcriptional profiles of mSDH GIST models recapitulate succinate dehydrogenase deficiency.

A. Scatterplots comparing gene expression levels (RNA sequencing) between patient tumors (x-axis) and mSDH GIST models (y-axis) as measured by Pearson correlation coefficient (r²>0.9). B. The relative expression levels (z-scores) for each gene comprising SDH loss related gene set (40) are indicated for each SDH-deficient tumor and models, as well as one WT SDH (non-SDH deficient) cell line (GIST882). C. Immunoblot of HIF-1α and HIF-2α proteins in KIT-mutant/SDH-WT (wildtype) GIST882 cells and in mSDH GIST models. D-G. qPCR analysis of HIF1A target gene VEGFA (D), LDHA (E), SLC2A1 (F) and SLC2A3 (G) expression in mSDH GIST models. Results are depicted as fold-change relative to WT-SDH (GIST882) cells. One-way ANOVA was performed for statistical analysis with P>0.05 (ns); *, P<0.05; ****, P<0.0001. H. The relative expression levels (z-scores) for each gene comprising hypoxia related gene set (43) are indicated for each SDH-deficient tumor and models, as well as one WT SDH (non-SDH deficient) cell line (GIST882).

Figure 4.. Temozolomide reduces cell viability of mSDH GIST models by induction of DNA double-strand breaks and impairing DNA repair.

A-C. Cell viability of mSDH GIST models determined by CellTiter-Glo viability assay after treatment by Imatinib (A), sunitinib (B) and temozolomide (C). Viability was measured after 3 days of treatment for imatinib and sunitinib and on 7 days of TMZ treatment. Data are presented as mean ± s.d. D. Representative images of a neutral comet assay for mSDH GIST models treated with either DMSO or 500 μM TMZ for 72 h. Comet tail lengths were measured (n = 100+ cells per group) and plotted. E. Representative immunofluorescence images of mSDH GIST models treated with DMSO or TMZ (500 μM) and stained for γ-H2AX⁺ nuclei. Quantification of γ-H2AX ⁺ cells in mSDH GIST models treated with DMSO or TMZ for 72 h. For each GIST model, γ-H2AX ⁺ nuclei were quantified and shown as % of total nuclei. Mann-Whitney t-test was performed for statistical analysis with **, P<0.01; ***, P<0.001, ****, P<0.0001. F. Immunoblots of mSDH GIST models treated with DMSO or TMZ (500 μM) for 3 days and probed for indicated proteins. G. Immunoblots of mSDH GIST models treated with DMSO or TMZ (500 μM) for 7 days and probed for indicated proteins. CC3 denotes cleaved caspase 3.

Figure 5.. Human SDH-mutant GISTs are sensitive to TMZ.

A-D. CT scan of a 22-year old male (Patient 5) with SDHB-mutant GIST before (A) and after 9 months of TMZ (B). PET scan of a 31-year old male (Patient 4) with SDHB-mutant GIST before (C) and after 2 cycles of TMZ (D). Red arrows indicate representative examples of tumor responses to TMZ. E. Waterfall plot demonstrating best tumor responses in 5 SDH-mutant GIST patients treated with TMZ. F. Swimmer’s plot demonstrating partial response, stable disease, and progressive disease in the same SDH-mutant GIST patients treated with TMZ depicted in 5E. G-H. Kaplan-Meier survival curves for SDH-deficient GIST patients treated with TMZ. Overall survival (OS) from date of diagnosis (G) and OS from start of TMZ treatment (H).

Figure 6.. Characteristics of mSDH models and proposed mechanism of action of TMZ.

Schematic representation highlighting metabolic and transcriptional characteristics of SDH-deficient parental tumors recapitulated by our patient-derived mSDH GIST models. Mechanism of action for TMZ proposed is also depicted. TMZ induces DNA damage, reduces the MGMT levels and leads to apoptosis. Created with BioRender.com.