Role of Vav1 Genomic Alterations in T-Cell Differentiation and Transformation in Peripheral T-Cell Lymphoma

Peripheral T-cell lymphomas (PTCL) are highly aggressive, malignant hematologic tumors that arise from clonal proliferation of mature post-thymic T-cells. Among these, angioimmunoblastic T-cell lymphoma (AITL), and peripheral T-cell lymphomas not otherwise specified (PTCL, NOS) account for >45% diagnoses, show limited response to intensified chemotherapy treatment with dismal overall survival rate. Our genomic studies have uncovered recurrent mutations and novel cancer-associated gene fusions involving the guanine nucleotide exchange factor VAV1 in AITL and PTCL, NOS, supporting a role for the VAV1 oncogene in the pathogenesis of PTCL. Genetic alterations in VAV1 found in PTCL included recurrent deletions resulting in the in-frame deletion of 9 codons (DV778_T786) in the linker region connecting the C-terminal SH2 domain to the downstream SH3 domain on VAV1 as well as recurrent different gene-fusion transcripts encoding proteins in which the C-terminal SH3 domain of VAV1 is replaced by different domains of the fusion partner. Consistent with the auto-inhibitory role of the C-SH3 domain, the PTCL-associated VAV1 alterations resulted in increased VAV1 activity and strong activation of the mitogen-activated protein kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and nuclear factor of activated T-cells (NFAT) pathways. Interestingly, mutation co-occurrence analysis shows significant mutual exclusivity of the highly prevalent AITL RHOA G17V mutation and VAV1 genomic alterations (p-value 0.0142) in AITL and PTCL, NOS. This observation supports that VAV1 genetic alterations and RHOA G17V could share a common mechanism of action.

To study the role and mechanisms of VAV1 alterations in PTCL in vivo we have engineered a C57/BL6 mouse lines with conditional expression of the recurrent Vav1-Myo1f fusion. By crossing Vav1-myo1f conditional knockin with CD4-CreER^T2 mouse deletor line, we have generated animals with CD4⁺ T cell-specific and tamoxifen-inducible expression of Vav1-myo1f. Preliminary studies indicate that expression of Vav1-myo1f in CD4⁺ cells induces expansion of TFH cells. This phenotype partially resembles that of animals with CD4 specific expression of Rhoa G17V, indicating a potential convergent effect and mechanisms for VAV1 alterations and RHOA G17V in T-cell development and transformation. However, and more interestingly, Vav1-myo1f expressing cells specifically present a memory cell-associated immunophenotype (CD44⁺ CD62L^-) and characteristic Th2-like features that had not been observed in the Rhoa G17V model. Analysis of signaling pathways activated in control and Vav1-myo1f expressing CD4⁺ cells showed increased phosphorylation of Vav1 and increased activation of the MAPK pathway upon expression of Vav1-myo1f, supporting the role of Vav1 alterations as regulators of T-cell activation. Notably, single cell transcriptome analysis of bulk splenic cells isolated from CD4-CreER^T2 control and CD4-CreER^T2Vav1^{co-Vav1-myo1f/WT} mice indicate that expression of Vav1-myo1f in CD4⁺ cells leads to a significant expansion of monocytes and TFH cells and a decrease in B-cell, both naïve and memory. These results support that expression of Vav1-myo1f in CD4⁺ cells can deeply modify the lymphoid microenvironment. Finally, preliminary analysis of a limited number of mice with targeted expression of the Vav1-Myo1f fusion in CD4⁺ cells (CD4-Cre Vav1^{co-Vav1-Myo1f /WT}) showed development of a fatal CD4⁺ malignant lymphoproliferation with a latency of 6-14 months. Histological examination showed disrupted splenic architecture accompanied with clonal expansion of CD4⁺ cells indicative of T-cell lymphoma with PTCL, NOS characteristics and verified expression of both wild type Vav1 and Vav1-myo1f alleles. Interestingly, Vav1-myo1f lymphoma cells showed Th2 associated features including expression of Gata3, c-Maf and IL4 and Il10.

These results support an important role for VAV1 alterations in T-cell differentiation, remodeling of the tumor microenvironment and T-cell lymphoma development and highlight the importance of our model as a new experimental system to study the evolution of PTCL and develop novel targeted therapies for this disease.