Preclinical Modeling of Leiomyosarcoma Identifies Susceptibility to Transcriptional CDK Inhibitors through Antagonism of E2F-Driven Oncogenic Gene Expression

Abstract

Purpose: Leiomyosarcoma (LMS) is a neoplasm characterized by smooth muscle differentiation, complex copy-number alterations, tumor suppressor loss, and the absence of recurrent driver mutations. Clinical management for advanced disease relies on the use of empiric cytotoxic chemotherapy with limited activity, and novel targeted therapies supported by preclinical research on LMS biology are urgently needed. A lack of fidelity of established LMS cell lines to their mesenchymal neoplasm of origin has limited translational understanding of this disease, and few other preclinical models have been established. Here, we characterize patient-derived xenograft (PDX) models of LMS, assessing fidelity to their tumors of origin and performing preclinical evaluation of candidate therapies.

Experimental design: We implanted 49 LMS surgical samples into immunocompromised mice. Engrafting tumors were characterized by histology, targeted next-generation sequencing, RNA sequencing, and ultra-low passage whole-genome sequencing. Candidate therapies were selected based on prior evidence of pathway activation or high-throughput dynamic BH3 profiling.

Results: We show that LMS PDX maintain the histologic appearance, copy-number alterations, and transcriptional program of their parental tumors across multiple xenograft passages. Transcriptionally, LMS PDX cocluster with paired LMS patient-derived samples and differ primarily in host-related immunologic and microenvironment signatures. We identify susceptibility of LMS PDX to transcriptional cyclin-dependent kinase (CDK) inhibition, which disrupts an E2F-driven oncogenic transcriptional program and inhibits tumor growth.

Conclusions: Our results establish LMS PDX as valuable preclinical models and identify strategies to discover novel vulnerabilities in this disease. These data support the clinical assessment of transcriptional CDK inhibitors as a therapeutic strategy for patients with LMS.

Figure 1.. LMS PDX maintain histologic and copy number changes across generations.

A, Mitotoses per 10 high-power fields (HPF) comparing parental tumors of non-engrafting and engrafting PDX. Data were compared by two-tailed t-test; **P = 0.0011. B, Engraftment rates comparing parental tumors of high- or low/intermediate-grade. C-D, H&E, smooth muscle actin (SMA) and desmin (DES) staining of the parental tumor and serial passages of LMS7 (C, intermediate-grade) and LMS20 (D, high-grade); scale bar = 50 μm. Inset images in D indicate focal and weakly desmin-positive cells. E, Copy number plots generated from ULP-WGS in the LMS33 parental tumor and subsequent generations of PDX extending to F17. The x-axis indicates chromosome and y-axis copy number (log₂ ratio). F, Copy number plots generated from ULP-WGS of plasma derived from LMS33 to detect circulating tumor DNA (ctDNA).

Figure 2.. Transcriptional profiling of LMS tumors and preclinical models.

A, Unsupervised hierarchical clustering of RNA-seq data from the 10,000 highest expressed genes (rows) across LMS patient samples, early and late PDX passage and cell lines (columns). * indicates one PDX that failed to co-cluster with its parental tumor. Patient samples were included in this analysis which did not have an associated engrafted PDX. B, PCA of LMS patient samples (red), early and late PDX (blue and yellow, respectively) and cell lines (gray). C-E, Plot of Log₂ FPKM in primary tumors and early and late generations of PDX for genes associated with molecular subtypes of LMS including SYNM and ADIRF (cLMS, black), PDGFRA and DCN (iLMS, blue), and ESR1 and CHRDL2 (uLMS, red). F, GSEA butterfly plot of false discovery rate (FDR) and normalized enrichment score (NES) for the C7: Immunologic Signatures gene sets. The Hallmark (H) gene sets for Angiogenesis, Hypoxia, Inflammatory Response, Interferon Gamma Response, Interferon Alpha Response, Allograft Rejection, Complement, IL6-JAK-STAT3 Signaling, TNFA Signaling via NFKB and IL2-STAT5 Signaling are also shown. G-H, GSEA plots of Hallmark gene sets for Inflammatory Response and Hypoxia.

Figure 3.. Failure of ATR and IGF1R inhibitors to perturb LMS PDX growth.

A, Expression in FPKM of ATRX across the indicated PDX lines. Data were analyzed by one-way ANOVA with Dunnet multiple comparisons test (compared to LMS33; ***,P<0.001). B, Tumor volume of LMS33 PDX in response to treatment with vehicle (n = 7) or ATR inhibitor berzosertib (60 mg/kg by oral gavage, 5 days per week; n = 5). C, Expression in FPKM of IGF1R across the indicated PDX lines. Data were analyzed by one-way ANOVA with Dunnet multiple comparisons test (compared to LMS20; ***,P<0.001). D, ISH of IGF1R in LMS19 and LMS20 parental tumors and PDX; scale bar = 5 μm. E, Tumor volume of LMS20 PDX in response to treatment with vehicle (n = 3) or IGF1R inhibitor linsitinib (50 mg/kg by oral gavage, 5 days per week; n = 4).

Figure 4.. HT-DBP of LMS patient samples and PDX identifies sensitivity to transcriptional CDK inhibitors.

A, Heatmap showing delta priming from HT-DBP of four LMS PDX and two patient samples (n = 2 per data point). Compounds used in the screen are organized into rows by drug class (Table S2). B, Cytochrome c release assay in LMS33 utilizing the indicated concentrations of CDK inhibitors, with DMSO, doxorubicin and navitoclax used as controls (n = 6). C, Tumor volume of LMS33 PDX in response to treatment with vehicle (n = 7) or flavopiridol (50 mg/kg i.p., 5 days per week; n = 6). Data were analyzed by two-way ANOVA, compared to vehicle; ***,P<0.001. D, Representative images of LMS33 treated with flavopiridol or vehicle for 3 days and stained for Ki-67 (scale bar = 50 μm) or CC3 (scale bar = 100 μm). E, Quantification of Ki-67-positive cells in LMS33 PDX treated with vehicle (n = 4) or flavopiridol (n = 3) for 3 days. Data were compared by two-tailed t-test; *,P<0.05.

Figure 5.. Selective CDK7 inhibition decreases tumor growth and the E2F-driven oncogenic program in LMS.

A, Tumor volume of LMS4 PDX in response to treatment with vehicle (n = 5) or YKL-5–124 (2.5 mg/kg i.p., 5 days per week; n = 5). B, Tumor volume of LMS33 PDX in response to treatment with vehicle (n = 7) or YKL-5–124 (2.5 mg/kg i.p., 5 days per week; n = 6). Data were analyzed by two-way ANOVA, compared to vehicle; ***,P<0.001. C, GSEA butterfly plot of FDR and NES for RNA-seq data comparing LMS33 tumor-bearing mice treated with vehicle (n = 3) or YKL-5–124 (n = 3) for 5 days. The top three gene sets are labeled in each condition. D-F, GSEA plots of Hallmark gene sets for Epithelial Mesenchymal Transition (EMT), E2F Targets and G2M Checkpoint; the NES and FDR are shown. G, Expression in FPKM of exemplary genes downregulated by YKL-5–124 and involved in oncogenic gene transcription and cell division. H, Expression in FPKM of exemplary genes upregulated by YKL-5–124 and involved in gene transcription, cellular stress response and DNA repair. Data were compared by two-tailed t-test; *,P<0.05; **,P<0.01; ***,P<0.001.