ABSTRACT: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a para-caspase required for B-cell receptor (BCR)-mediated NF-κB activation. We sought to examine the role of MALT1 in B-ALL and determine the biological consequences of inhibiting its activity. First, we tested MALT1 expression by immunoblot in B-cell acute lymphoblastic leukemia (B-ALL) using 16 cell lines representing the disease spectrum and found that MALT1 was expressed in all cell lines at different levels. To determine sensitivity to MALT1 inhibition we used two molecules: Z-VRPR-fmk, a highly selective MALT1 blocking peptide, and MI2, a small molecule MALT1 inhibitor. Z-VRPR-fmk resulted in a dramatic cell growth inhibition in most of our B-ALL cell lines, with appropriate positive (JEKO) and negative (JURKAT) controls. We did not observe a clear correlation between MALT1 level and degree of sensitivity to Z-VRPR-fmk. Interestingly, the two ibrutinib-resistant cell lines RS4;11 and 697, were amongst the top sensitive cell lines to MALT1 inhibition. A similar pattern of cell sensitivity was observed when these cell lines were treated with MI2, with a positive correlation between the IC50 of MI2 and the amount of cell growth inhibition with Z-VRPR-fmk (r=0.74 95%CI (0.47-0.88) p=0.0001). Interestingly, freshly collected PBMCs from patients with B-ALL showed a high sensitivity to MI2 following incubation at serial dilutions for 48h. Furthermore, apoptotic and cell cycle analyses showed a dose-dependent apoptotic induction and cell cycle arrest in the studied ell lines. Surprisingly, the proteolytic activity of MALT1, studied by measuring its ability to cleave its targets such as CYLD, BCL10, and RelB, was not detected at baseline, nor after proteasomal inhibition with MG-132 or following crosslinking of pre-BCR with anti-IgM in pre-B ALL, the latter successfully increased AKT phosphorylation. Collectively, these data are suggestive of distinct role for MALT1 in B-ALL. To explore this possibility, we used RNA sequencing to determine the changes in gene expression profiling following an 8h MI2 treatment in 12 sensitive B-ALL cell lines. Out of 39,514 tested genes, there were 160 genes whose expression changed ≥ 2-fold at P < 0.05 (84 down- and 76 up-regulated). Gene Set Enrichment Analysis (GSEA) identified 34 Hallmark and Oncogenic Signature gene sets relevant to B-ALL that were all downregulated by MI2 (FDR < 10%, and normalized enrichment score (NES) ≥ 1.50). Among those gene sets the c-MYC, gene signatures stood out as the most affected, further confirmed with downregulation of the MYC protein by immunoblot analysis in MI2-sensitive cell lines. In conclusion, MALT1 plays a critical role in B-ALL survival likely through a novel mechanism that involves MYC regulation, independently from pre-BCR/BCR signaling.
