Beyond MYD88: Implications of TNFAIP3 Loss on MYD88 Driven Lymphomas

MYD88 mutations are one of the most recurrent mutations in hematologic malignancies and are found in 19% of lymphomas. While it is detected in many subtypes of B-cell malignancies, its prevalence is highest in Waldenstrom Macroglobulinemia (90-100%, WM) and the ABC subtype of diffuse large B-cell lymphoma (39%, DLBCL). While the biologic impact of MYD88_L265P has been well-studied, little is known about its function in the context of a complex genetic background, which is often seen in DLBCL. Recent mouse models suggest that MYD88_L265P alone may not be sufficient to induce tumor formation and that the pathway requires additional genetic hits. Interplay between MYD88_L265P and other oncogenic events are further supported by the fact that TNFAIP3 (A20) inactivation often accompanies MYD88_L265P . This data suggests a potential relationship between these genetic events, and more importantly, a possible clinical impact. The aim of this study was to determine the clinical impact of MYD88_L265P and TNFAIP3 loss and to generate a human cell line model that mimics the effect of MYD88_L265P / TNFAIP3 loss in hopes to understand the cellular consequence of these genomic alterations and identify therapeutic targets.

MYD88 mutations have been reported to associate with a poor outcome in DLBCL and upon review of our whole exome sequencing and clinical data (n=147) we found that mutations in MY D88 (n=20) are commonly found in combination with TNFAIP3 loss (n=11, 55%). Additionally, review of RNASeq data (n=33) reveals that the MYD88 and NF-κB target genes IL-6 and BCL2 are elevated (1.4 fold and 1.5 fold) in patients with TNFAIP3 loss (n=7). To better understand the impact of a TNFAIP3 loss in combination with the MYD88_L265P mutation,we used the WM cell line MWCL1 and the ABC-DLBCL cell line HBL-1, both of which harbor a heterozygous mutation in MYD88_L265P . For the TNFAIP3 specific knock out, we used the transcription activator-like effector nucleases (TALENs) genome editing approach to induce a double stranded break in exon 5 of the TNFAIP3 gene. TALENs were expressed in lymphoma cells by transient transfection, and single-cell clones were isolated. Sanger sequencing of the TNFAIP3 gene was performed to identify clones that carried the deletion and loss of A20 was confirmed by Western blot. We then explored the impact of TNFAIP3 loss on MYD88_L265P-driven p38 and NF-κB activation and found that phosphorylation of p38 and NF-κB is significantly higher in the MWCL1 and HBL-1 A20 knockout cell lines (p≤ 0.05). We next looked at expression of NF-κB target genes by quantitative real time PCR and found that IL-6 and CXCL-10 (IP-10) were significantly upregulated in the MWCL1 and HBL-1 A20 knockout cell lines compared to their wild type cell lines (p≤ 0.05). Upregulation of IL-6 and CXCL-10 protein expression was confirmed by Luminex. Because of the increased cytokine expression detected in A20 knockout cells, we next looked at JAK/STAT signaling proteins and found that phosphorylation of STAT3 was higher in MWCL1 and HBL-1 A20 knockout cells.

In summary, we have established new WM and DLBCL cell line models that mimic the effect of the MYD88_L265P mutation in combination with a loss of the TNFAIP3 gene and A20 expression. We show that loss of TNFAIP3 results in a higher baseline phosphorylation of NF-κB, p38 and STAT3. Additionally, loss of TNFAIP3 impacts expression of IL-6 and IP-10. Overall, results from this study contribute to our understanding of MYD88 driven lymphomas, suggests a possible clinical implication for those individuals that harbor both a MYD88 mutation and a loss of TNFAIP3, and also provides us with a useful model to study novel therapeutic strategies in patients who harbor these genetic variants.