Modeling Extraordinary Response Through Targeting Secondary Alterations in Fusion-Associated Sarcoma

Abstract

Purpose: Targeted therapy in translocation-associated sarcomas has been limited to oncogenic activation of tyrosine kinases or ligands while gene fusions resulting in aberrant expression of transcription factors have been notoriously difficult to target. Moreover, secondary genetic alterations in sarcomas driven by translocations are uncommon, comprising mostly alterations in tumor suppressor genes (TP53, CDKN2A/B). Our study was triggered by an index patient showing a dramatic clinical response by targeting the secondary BRAF V600E mutation in a metastatic angiomatoid fibrous histiocytoma (AFH) harboring the typical EWSR1::CREB1 fusion.

Materials and methods: The patient, a 28-year-old female, was diagnosed with an AFH of the thigh and followed a highly aggressive clinical course, with rapid multifocal local recurrence within a year and widespread distant metastases (adrenal, bone, liver, lung). The tumor showed characteristic morphologic features, with histiocytoid cells intermixed with hemorrhagic cystic spaces and lymphoid aggregates. In addition to the pathognomonic EWSR1::CREB1 fusion, targeted DNA sequencing revealed in both primary and adrenal metastatic sites a hot spot BRAF V600E mutation and a CDKN2A/B deletion. Accordingly, the patient was treated with a BRAF-MEK inhibitor combination (encorafenib/binimetinib) showing an excellent but short-lived response.

Results: Using a CRISPR-Cas9 approach, we introduced the BRAF c.1799 T>A point mutation in human embryonic stem (hES) cells harboring a conditional EWSR1 (exon7)::CREB1 (exon7) translocation and further differentiated to mesenchymal progenitors (hES-MP) before fusion expression. The cells maintained the fusion transcript expression and the AFH core gene signature while responding to treatment with encorafenib and binimetinib.

Conclusion: These results highlight that additional targeted DNA NGS in chemotherapy-resistant translocation-associated sarcomas may reveal actionable oncogenic drivers occurring as secondary genetic events during disease progression.

Appendix Figure 1.. Strategy for generating BRAF V600E mutation in hES cells and screening method.

(A) BRAF gene diagram (exon 15), highlighting the sgRNA (green box) and PAM (purple box) sequences. The Alu-I cutting site and codon for Valine amino acid are underlined. After HDR integration of ssDNA carrying the 2 mutant nucleotides (red), the Alu-I cutting site is lost, while the codon for the valine residue is substituted with the codon for glutamic acid. (B) Schematic representation of the PCR strategies for screening the BRAF V600E mutant. In the allele specific PCR, the primer pair is composed of a universal primer and a mutation-specific primer, which will amplify the mutant alleles. The positive clones were confirmed by an amplification across the targeted site and PCR product digestion with Alu-I. (C) Western blot with BRAF VE1 antibody on the hES BRAF V600E mutant clone used for experiments presented in Figs. 3 and 4. (D) EWSR1::CREB1 fusion RT-PCR showing comparable transcript levels in the BRAF V600E-mutant hES cells compared to the isogenic cell line.

Appendix Figure 2.. Time course for EWSR1::CREB1 fusion and AFH core gene signature expression in Cre-transfected cells after sorting.

(A) RT-PCR and (B) qRT-PCR as described in Figure 3. BRAF V600E-mutant mesenchymal cells and isogenic cell lines transfected with a plasmid expressing the Cre-recombinase and an mCherry fluorescent marker. Three days after transfection, mCherry positive cells were sorted and replated for time course experiment and analysis. Histograms represent the fold increase compared to -Cre condition at day 6 of 1 experiment.

Appendix Figure 3.. RNAseq analysis on hES-MP BRAF V600E cells expressing the EWSR1-CREB1 fusion.

(A) Histogram plots showing upregulation of PTPRN, PLAT, VCAN and TGFBI in BRAF V600E mutant cells expressing the EWSR1::CREB1 fusion compared to cells expressing fusion alone and (B) GSEA on deregulated genes from the same analysis.

Figure 1.. Radiologic imaging of local recurrence and adrenal metastasis pre- and post-therapy with encorafenib and binimetinib combination.

Upper panels show MRI of the right femur showing 2^nd local recurrence (asterisk) with fluid-fluid level (left); large increase in size of the primary tumor mass in the thigh over one month period (center); and significant decrease in size after targeted therapy (right). Lower panels show CT scans for the same timepoints, with small adrenal gland metastasis (asterisk, left), with large increase in size (center) and subsequent decrease in size after targeted therapy (right).

Figure 2.. Pathologic features and molecular findings.

(A) Histologic images from the 2^nd local recurrence showing at low power (left) a partly cystic-hemorrhagic and partly solid mass surrounded by a prominent rim of lymphoid aggregates. At higher magnification (center) the tumor is composed of sheets of plump histiocytoid cells with pale eosinophilic cytoplasm and reniform nuclei with open chromatin and mild atypia. Immunohistochemical stain with BRAF VE1 antibody shows diffuse, strong cytoplasmic positivity in the tumor cells (right). (B) Archer FusionPlex showed the presence of a fusion transcript composed of EWSR1 exon7 fused to CREB1 exon7. (C) IMPACT testing from the first local recurrence and the adrenal metastasis showed clonal BRAF V600E mutation and a CDKN2A/B deletion. The EWSR1::CREB1 fusion was also identified. The adrenal metastasis showed in addition a PAK7 mutation.

Figure 3.. EWSR1::CREB1 fusion expression and AFH core gene signature in BRAF V600E mutant mesenchymal cells.

(A) RT-PCR for EWSR1::CREB1 transcript and (B) qRT-PCR for AFH signature genes in a time course experiment after transduction with Cre recombinase in mesenchymal cells harboring the BRAF V600E mutation. Histograms represent the fold increase compared to -Cre condition at day 5 from 3 independent experiments. Statistical significance is calculated with a paired t-test comparing +Cre with the corresponding -Cre condition. *p<0.05, **p<0.01; when not indicated the difference is not statistically significant.

Figure 4.. Viability and induction of apoptosis subsequent to encorafenib and binimetinib treatment in BRAF V600E mutant mesenchymal cells expressing EWSR1::CREB1 fusion protein.

(A) Cell viability of BRAF V600E mutant cells and parental cells after 7-day continuous treatment with increasing concentrations of encorafenib or binimetinib. (B) Cell viability after 7-day treatment with encorafenib (250 nM) and binimetinib (25 nM) in mesenchymal cells prior to induction of the EWSR1::CREB1 fusion. (C) Cell viability in a pool of Cre-transfected mesenchymal cells after 7-day treatment with encorafenib or binimetinib, at day 5 or day 12 post-transfection (e.v. empty vector). (D) Annexin V measurement of apoptosis in cells transfected with the Cre recombinase after 72-hour treatment with single dose of encorafenib, binimetinib or both. Each panel error bar represents the standard deviation from at least 3 independent experiments. For panel B and D unpaired t test * p< 0.05, ** p < 0.01, if not indicated the difference was not significant.