ID3 Loss In Vivo Synergizes with MYC-Driven Oncogenesis in Burkitt Lymphoma

Burkitt lymphoma (BL) is an aggressive cancer, characterized by the deregulation of the c-myc (MYC) gene. BL is a major model disease for molecular studies in many other malignancies. Treatment of BL requires long courses of intensive chemotherapy which extracts a disproportionate toll on patients.

Previous studies using next generation sequencing have identified ID3 as a tumor suppressor gene, with silencing mutations in about 40% of BLs. However, relatively little is known regarding the biological role of ID3 in BLs.

In this study, we sought to elucidate the molecular mechanisms that are affected by ID3 loss in BL. We utilized two complementary strategies. First, we developed an in vivo approach through conditional knockout of Id3 in mice. Second, we utilized a CRISPR-based approach to genetically knockout ID3, to study the effects of ID3 loss in BL cell lines.

We have successfully knocked out exon 1, including the functional helix-loop-helix (HLH) domain of ID3 in 4 BL cell lines (Namalwa, Ramos, BJAB, and Jijoye) that do not harbor mutations in ID3. Deletion of exon 1 causes complete deletion at the protein level. Transcriptome sequencing from ID3 mutated BL cases identified increase in the expression of MYC target genes, as well as proliferation-related genes. Cell cycle analyses revealed faster cell cycle progression in the ID3 knockdown cells (P=0.02).

To study ID3 loss in vivo, we crossed Em-Myc+ mice with a standard Id3 knockout strain mice. We found that E m -Myc⁺;Id3^-/+ mice demonstrated reduced latency to tumor development compared to Eµ-Myc⁺;Id3^+/+ littermates (P<0.01). This decreased tumor latency noted in the E m -Myc⁺;Id3^-/+ mice suggests a strong in vivo synergy between the oncogenic effects of MYC overexpression and ID3 loss. Many of these tumors displayed the classic "starry sky" pattern that is classic for human Burkitt lymphomas. We are continuing to use the AID -Cre positive strains to generate combined Id3 deletion and Myc over-expression specifically in germinal center B cell context. Additional characterization of the mouse tumors using mouse whole exome sequencing and transcriptome sequencing is in progress and will be presented in the meeting.

Our data reveal that ID3 greatly potentiates the oncogenic role of the MYC gene and understanding the role of ID3 enables new insights into the pathogenesis of BL, and potentially other MYC-driven malignancies.