Deletions of 6q15-16.1 are recurrently found in pediatric T-cell acute lymphoblastic leukemia (T-ALL). This chromosomal region includes the mitogen-activated protein kinase kinase kinase 7 (MAP3K7) gene which is crucial for innate immune signaling. It is functionally and prognostically relevant both in hematologic malignancies and solid tumors. However, its distinct biological functions and relevance for different tumor entities are cell-type specific and remain controversial. Here, we show that MAP3K7 is recurrently deleted in pediatric T-ALL cases and that a residual expression of MAP3K7 is indispensable for T-ALL cell lines.
We determined the frequency of MAP3K7 deletions in a cohort of 327 pediatric T-ALL patients by multiplex ligation-dependent probe amplification (MLPA). In a subgroup, we additionally employed targeted sequencing to identify point mutations. Deletions of MAP3K7 were identified in approximately 10% and point mutations in approximately 1% of children with T-ALL. All deletions of 6q15 were heterozygous and the adjacent CASP8AP2 gene was co-deleted in 32 of 33 cases. Clinically, the MAP3K7/CASP8AP2 deletions were significantly associated with a mature T-ALL immunophenotype, but not with any other patient characteristics or with the cumulative incidence of relapse and overall survival. Furthermore, deletions of MAP3K7/CASP8AP2 were preferentially associated with SIL-TAL1 fusions. Within the subgroup of SIL-TAL1 positive patients, those with MAP3K7/CASP8AP2 deletion showed a trend towards a higher risk of relapse compared to SIL-TAL1 -positive MAP3K7/CASP8AP2 -negative patients (25% vs 8%, p=0.13).
We next analyzed the effects of shRNA-mediated MAP3K7 depletion on cell proliferation, apoptosis and NF-κB activation in three T-ALL cell lines (CCRF-CEM, Jurkat, MOLT4) that do not carry MAP3K7 deletions. We aimed at phenocopying a MAP3K7 deletion by using an Adeno-associated viral vector-mediated transduction system to efficiently deliver anti-MAP3K7 shRNA together with a GFP reporter to these cell lines. Transduction efficiency was above 80% for the cell lines CCRF-CEM and Jurkat. Depletion efficiency was uniformly in the range of 75%. Treatment with anti-MAP3K7 shRNA in comparison to non-silencing shRNA significantly slowed down proliferation in all three T-ALL cell lines and resulted in an increase of apoptotic cells up to 8-fold as measured by Annexin V staining.
In order to assess the contribution of NF-κB signaling to the effects of MAP3K7 depletion, cells were treated with TNF-α and cell lysates analyzed for components of the NF-κB pathway by Western blotting and for expression of the NF-κB target genes BCL2, CMYC, FAS, PTEN and TNF-α by RT-PCR. Stimulation with TNF-α led to rapid and transient degradation of IκB and an accumulation of phospho-NF-κB p65 (Ser536), while other NF-κB proteins (p100, p105, p50) remained unaffected. Depletion of MAP3K7 neither changed IκB levels in unstimulated cells nor did it prevent the degradation of IκB after stimulation with TNF-α. Furthermore mRNA expression of none of the NF-κB target genes was consistently changed by more than 2-fold in any of the cell lines after MAP3K7 depletion. We conclude that in T-ALL cell lines, MAP3K7 is not required for the degradation of IκB and subsequent activation of NF-κB after stimulation with TNF-α.
This study revealed that the recurrent deletion of MAP3K7/CASP8AP2 is associated with SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and risk of relapse. Homozygous deletions of MAP3K7 were not observed, and efficient depletion of MAP3K7 interfered with viability of T-ALL cells, indicating that a residual expression of MAP3K7 is indispensable for T-lymphoblasts.
Schrappe: Medac: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.
Asterisk with author names denotes non-ASH members.