Integrated mate-pair and RNA sequencing identifies novel, targetable gene fusions in peripheral T-cell lymphoma

Abstract

Peripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of T-cell malignancies that generally demonstrate aggressive clinical behavior, often are refractory to standard therapy, and remain significantly understudied. The most common World Health Organization subtype is PTCL, not otherwise specified (NOS), essentially a "wastebasket" category because of inadequate understanding to assign cases to a more specific diagnostic entity. Identification of novel fusion genes has contributed significantly to improving the classification, biologic understanding, and therapeutic targeting of PTCLs. Here, we integrated mate-pair DNA and RNA next-generation sequencing to identify chromosomal rearrangements encoding expressed fusion transcripts in PTCL, NOS. Two of 11 cases had novel fusions involving VAV1, encoding a truncated form of the VAV1 guanine nucleotide exchange factor important in T-cell receptor signaling. Fluorescence in situ hybridization studies identified VAV1 rearrangements in 10 of 148 PTCLs (7%). These were observed exclusively in PTCL, NOS (11%) and anaplastic large cell lymphoma (11%). In vitro, ectopic expression of a VAV1 fusion promoted cell growth and migration in a RAC1-dependent manner. This growth was inhibited by azathioprine, a clinically available RAC1 inhibitor. We also identified novel kinase gene fusions, ITK-FER and IKZF2-ERBB4, as candidate therapeutic targets that show similarities to known recurrent oncogenic ITK-SYK fusions and ERBB4 transcript variants in PTCLs, respectively. Additional novel and potentially clinically relevant fusions also were discovered. Together, these findings identify VAV1 fusions as recurrent and targetable events in PTCLs and highlight the potential for clinical sequencing to guide individualized therapy approaches for this group of aggressive malignancies.

Figure 1

Distribution of 378 genomic events identified by mate-pair sequencing in 11 cases of PTCL, NOS. (A) Number of genomic events per case. (B-D) Representative Circos diagrams illustrating heterogeneity in the degree of complexity of genomic events among PTCLs: (B) high complexity; (C) focally high complexity with multiple rearrangements involving chromosomes 16 and 19 (chromothripsis); and (D) low complexity. (E) Distribution of intrachromosomal and interchromosomal events. (F) Distribution of events based on involvement of genes or nongenic regions and identification of expression fusion transcripts by RNA sequencing.

Figure 2

Identification and validation of VAV1 fusions in PTCL, NOS. (A) Rearrangement of VAV1 and GSS genes as detected in genomic DNA by MPseq. Vertical and oblique black lines represent aberrant read-pairs; blue ends indicate mapping to (+) strand, whereas red ends indicate mapping to (−) strand. (B) VAV1-GSS fusion transcript as detected by RNAseq. Bridging reads spanning the fusion site are shown. TSS, transcription start site. (C) Sanger sequencing of the fusion site in VAV1-GSS fusion. (D) Schematic diagrams of VAV1 protein domains in wild-type VAV1 and resulting from VAV1 fusions. (E) FISH confirming a VAV1 rearrangement in a tumor cell nucleus (blue) from a PTCL, NOS specimen. The normal intact VAV1 allele is indicated by a red-green fusion (f) signal. Disruption of the VAV1 gene region on the other allele is indicated by separation (s) into one red (5′) signal and 1 green (3′) signal. (F) Pathologic features of PTCL, NOS with VAV1 fusion (hematoxylin and eosin stain; inset, CD30 immunostain). Original magnification ×400 (inset, ×1000).

Figure 3

VAV1-GSS fusion drives cytoskeletal reorganization, migration, and proliferation. (A) VAV1-GSS fusion promotes cytoskeletal reorganization and (B) increased cell circularity in adherent NIH-3T3 cells. A circularity value of 1 indicates a perfect circle. (C) The VAV1-GSS fusion protein is phosphorylated at VAV1 Y174 in Jurkat cells. In Jurkat, VAV1-GSS drives cell (D) migration and (E) growth relative to both wild-type VAV1 and an empty vector control.

Figure 4

VAV1-GSS fusion induces targetable RAC1 dependence. (A) VAV1-GSS fusion induces Rac activation in Jurkat cells. (B) Rac activation data summarizing 3 independent experiments as shown in A. (C) RAC1 knock-down abrogates effect of VAV1-GSS fusion on cell growth. (D) Jurkat cells transfected with VAV1-GSS exhibit greater sensitivity to azathioprine than cells transfected with wild-type VAV1 or an empty vector control.

Figure 5

Novel ITK-FER kinase fusion in PTCL, NOS. (A) ITK-FER rearrangement as detected by MPseq. (B) ITK-FER fusion transcript as detected by RNAseq. (C) Sanger sequencing of the ITK-FER fusion site. (D) Exon-level RNAseq read plot for FER in TCL72 (“Case”) compared with the remaining 10 sequenced cases of PTCL, NOS. RPKM, reads per kilobase per million. (E) Schematic diagrams of protein domains in ITK, FER, and the ITK-FER fusion protein. (F) ITK-FER expression promotes colony formation in HEK-293 cells. PH, pleckstrin homology; TH, Tec homology; SH, Src homology; F-BAR, FER-CIP4 homology–Bin-amphiphysin-Rvs.