Splicing Factor 3B Subunit 1 (SF3B1) Mutations Impact FOXP1 Splicing in Chronic Lymphocytic Leukemia (CLL) Cells but Conserve the Balance Between Newly Described and Classical Splicing Variants of ZAP-70

Zeta-chain Associated Protein of 70 kDa (ZAP-70) is the second member of the Spleen tyrosine Kinase (Syk) family, and plays a key role in T-cell Receptor signalling. An alternative variant of ZAP-70 was described in 2004 by Hirokawa et al. producing a shorter protein named Truncated ZAP-70 Kinase (TZK). ZAP-70 has also been found expressed in some B-cell subsets and in various B-cell malignancies. In CLL, the expression of ZAP-70 retains a prognostic significance; however, its function is not completely understood. Recently, whole genome sequencing identified mutations affecting the spliceosome such as SF3B1 mutations that are associated with poor outcome in CLL. In this work, we aimed at identifying novel alternative transcripts of ZAP-70 in normal lymphocytes and in CLL cells. We also explored the potential link between ZAP70 alternative transcripts and SF3B1 mutations.

Peripheral blood mononuclear cells (PBMC) were obtained from normal subjects and CLL patients. T cells, NK cells and monocytes were purified by negative selection from 11 normal subjects. We explored the transcriptional profile of ZAP-70 by standard PCR using 4 combinations of primers spanning over the 14 exons of the transcript. PCR products were revealed by gel electrophoresis, purified and sequenced by Sanger method. The expression of ZAP-70 was studied by real-time quantitative PCR using SYBR Green® technique after designing specific primers for the four different transcripts individualized. The expression of the full length and truncated forms of FOXP1 was studied by real-time quantitative PCR as previously described (EHA2015 abst#4919). SF3B1 mutations were detected by Sanger sequencing of the exons 14, 15, and 16.

In Jurkat cell line, we identified two new variants of ZAP-70 corresponding to the alternative splicing of exon 3 and exons 3 and 4. We named them Δ3 and Δ3-4 respectively after confirmation by Sanger sequencing. To detect both Δ3 and Δ3-4 in the same reaction, we designed a PCR encompassing exons 1 to 5, and we were able to detect ZAP-70, Δ3 and Δ3-4 in peripheral blood unselected normal cells. SYBR Green ® technique with new sets of primers allowed us to explore ZAP-70, TZK and the two new variants in selected NK cells and T lymphocytes. As previously described, ZAP-70 was more expressed in NK cells than in T cells, and at lower level in CLL cells. TZK was also detected in T-lymphocytes and NK cells, Δ3 and Δ3-4 were found at very low levels in both cell types and were absent from ZAP-70 negative CLL cells. We studied ZAP-70 transcripts in 13 ZAP-70 positive CLL. All alternative transcripts were present with a pattern of expression different to that of T-cells. We have shown recently that in all cases of CLL with SF3B1 mutation, a high expression of the FOXP1 truncated variant was observed as compared to a very low level in unmutated samples. This variant was highly expressed in the samples carrying five different mutations of SF3B1 (Y623C, R625H, K700E, G740V and G742D). Among our 13 ZAP70 positive patients, 2 cases harboured the SF3B1 Y623C mutation. In these two cases, the balance between the four ZAP-70 transcripts detected showed no obvious difference with wild-type SF3B1 ZAP-70 positive cases.

In conclusion, we studied the transcription profile of ZAP-70 in various cells types and identified two novel alternative transcripts of ZAP-70 (Δ3 and Δ3-4). These transcripts were detected at various levels in all ZAP-70-positive cell types. Interestingly, we found that the presence of SF3B1 mutation impacted the splicing of FOXP1 transcription factor but not the balance between ZAP-70 transcripts We are currently expanding the cohort of ZAP-70 cases with other types of SF3B1 mutations. Mutations affecting the spliceosome such as SF3B1 mutations do not seem to impact transcription of ZAP-70.