The Paracaspase MALT1 Acts Independently of Pre-B-Cell Receptor Signaling As a Key Factor in Leukemic Cell Survival in Precursor B-Cell Acute Lymphoblastic Leukemia
Authors: Saba Nakhle S. , Meyers Jade , Fontan Lorena , Melnick Ari , Wiestner Adrian , Lobelle-Rich Patricia , Kim Ekaterina , Burger Jan A. , Mouawad Yara , Deininger Prescott L. , Socola Francisco , Safah Hana , Flemington Erik K 
Published: November 2019

Despite a central role for B-cell receptor precursor (pre-BCR) pathway in precursor B-cell acute lymphoblastic leukemia (B-ALL), there is limited available data on therapies that aim to disrupt this pathway. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a para-caspase required for BCR-mediated NF-κB activation. We recently showed that targeting MALT1 with the small molecule inhibitor MI2 is effective in CLL, including drug-resistant clones (Saba Can Res 2017). 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-ALL using 17 cell lines representing the disease spectrum (7 pro-B: REH, SEMK2, TOM1, RS4;11, NALM21, Z119, BV173; 8 pre-B: HB11;19, NALM6, RCH-ACV, SMS-SB, 697, KASUMI2, KOPN8, HPB-NULL; and 2 mature/Burkitt: 2F7, RAJI), 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 (TMD8) and negative (K562) controls, independent of the cell-of-origin (pro, pre, mature) or the presence of the Philadelphia chromosome. 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, resulting in an IC50 at 48h of 0.2 µM in RS4;11 and < 0.5 µM in other sensitive cell lines, which is consistent with published data in sensitive DLBCL cell lines (IC50, 0.2-0.5 µM), and our data on the CLL cell line MEC1 (IC50, 0.2 µM). We then tested freshly collected PBMCs from patients with various blood cancers presenting with a leukemic phase against serial dilutions of MI2 for 48h (7 B-ALL, 24 CLL, and 4 CML). In addition, we included normal B-cells collected from five volunteers. Interestingly, B-ALL samples showed the highest sensitivity to MI2, followed by CLL, while the rest were resistant.
The proteolytic activity of MALT1 can be studied by measuring its ability to cleave its targets such as A20, CYLD, BCL10, Roquin, Regnase and RelB. Surprisingly, with the exception of the Burkitt cell line 2F7, we did not detect cleavage of these targets 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. The constitutive activation of MALT1 in 2F7 was effectively inhibited by Z-VRPR-fmk as determined by a marked reduction in targets cleavage concomitant with an increase in full length proteins. We are expanding the mature B-ALL cell line cohort to include TANOUE, BALL-1, DAUDI, GA-10, and NC-37 cell lines to further explore to role of MALT1 in this disease subset. Collectively, these data highly suggest distinct roles for MALT1 in B-ALL: pro and pre-B-ALL vs. mature B-ALL.
To explore the possibility of distinct role for MALT1 in B-ALL, arguably independent of BTK and of signaling through BCR, we used RNA sequencing to determine the changes in gene expression profiling following a 24h treatment with Z-VRPR-fmk in 3 highly sensitive B-ALL cell lines (RS4;11, HPB-NULL, and 697). 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 Signatures gene sets relevant to B-ALL that were all downregulated by Z-VRPR-fmk (FDR < 10%, and normalized enrichment score (NES) ≥ 1.50). Among those gene sets, mTOR-S6K and TANK-binding kinase 1 (TBK1)-dependent gene signatures stood out as the most affected. MALT1 was shown to be involved in the ribosomal protein S6 phosphorylation through activation of mTOR/AKT signaling. Indeed, treatment with MI2 and Z-VRPR resulted in a significant decrease in S6 phosphorylation in RS4;11 and SEMK2.
In conclusion, MALT1 plays a critical role in B-ALL survival likely through a novel mechanism that involves mTOR-S6K pathway, independently from pre-BCR/BCR signaling.
Supported by a grant from the Ladies Leukemia League, Inc., of the Gulf South Region.

Disclosures
Saba: Kyowa Kirin: Consultancy; AbbVie: Consultancy; Janssen: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau. Melnick:Epizyme: Consultancy; Janssen: Research Funding; Constellation: Consultancy. Wiestner:Merck: Research Funding; Nurix: Research Funding; Pharmayclics: Research Funding; Acerta: Research Funding. Burger:AstraZeneca: Honoraria; Aptose Biosciences, Inc: Research Funding; Gilead Sciences: Research Funding; Janssen Pharmaceuticals: Consultancy, Honoraria; Pharmacyclics, an AbbVie company: Research Funding; BeiGene: Research Funding. Safah:Celgene: Speakers Bureau; Incyte: Speakers Bureau; Verastem: Honoraria, Speakers Bureau; Jazz: Speakers Bureau; Amgen: Honoraria, Speakers Bureau.