Abstract A19: MALT1 inhibition as an anchor for combinatorial therapy of ABC-DLBCL.
Authors: Fontan Lorena , Yang Chenghua , Shinglot Himaly , Kabaleeswaran Venkataraman , Laurent Volpon , Osborne Michael , Beltran Elena , Rosen Monica , Shaknovich Rita , Yang Shao N. , Gascoyne Randy D. , Cerchietti Leandro , MArtinez-Climent Jose A. , Glickman J Fraser , Borden KAtherine , Wu Hao , Melnick Ari 
Published: August 2015

MALT1 (Mucossa Associated Lymphoid tissue Lymphoma Translocated protein 1) is critical for the proliferation and survival of Activated B-cell like Diffuse Large B-cell Lymphoma (ABC-DLBCL), the most chemo-resistant form of DLBCL. MALT1 mediates activation of the B-cell receptor pathway (BCR) downstream of characteristic somatic mutations in CD79, CARD11 or MYD88 that lead to chronically activated NF-κB. MALT1 is a paracaspase and the effector enzyme of the CARD11/Bcl10/MALT1 signalosome, a high order assembly that functions as an amplifier of BCR signaling to NF-κB. MALT1 constitutes a compelling therapeutic target because: i) it is the only paracaspase in humans, ii) MALT1 knockout mice are viable, and iii) ABC-DLBCLs are biologically dependent on MALT1 paracaspase activity.

MALT1 is only active when forming multimeric complexes. In order to identify potential MALT1 inhibitors we engineered a leucine zipper-MALT1, obliged and enzymatically active dimer, and established a paracaspase enzymatic assay for high throughput screening. Screening a ~50,000 compound chemical diversity library allowed us the identification and validation of 19 distinct chemical scaffolds that inhibited MALT1 with an IC50&lt;20 μM. Three compounds significantly induced selective dose-dependent suppression of MALT1-dependent ABC-DLBCL cells (MI-2, p&lt;0.0001; MI-4, p=0.006; MI-11, p&lt;0.0001). The most potent compound in cell-based assays was MI-2 with a GI25 in the low nanomolar range. MI-2 analogs also displayed nanomolar activity. In depth analysis using NMR and LC-MS revealed that MI-2 binds covalently to the active site of MALT1. In DLBCL cells, MI-2 inhibited MALT1 cleavage of its targets TNFAIP3, BCL10 and RelB, as well as nuclear translocation of c-REL and overall NF-κB activation. MI-2 inhibited proliferation by inducing G1 arrest and ultimately promoted apoptosis in ABC-DLBCLs including those with mutations that bypass BTK inhibitors, like CARD11 activating mutation. MI-2 was non toxic in vivo and potently and specifically inhibited the growth of xenotransplanted ABC-DLBCLs (p=0.014, t-test) but not GCB-DLBCLs. Moreover, MI-2 selectively killed primary human ABC-DLBCL specimens ex vivo.

Given that multiple pathways contribute to ABC-DLBCL pathogenesis, we hypothesized that MALT1 inhibitors would be most effective within combinatorial therapy regimens. Along these lines MI-2 strongly enhanced the activity of CHOP chemotherapy drugs against ABC-DLBCL cells. The addition of MI-2 to doxorubicin allowed for 2.5 to 13-fold reduction in the doxorubicin dose as determined by the dose-reduction index that was specific for the doxorubicin resistant cell lines (GI50 &gt; 200 nM). Because the BCR pathway constitutes a complex network of signaling molecules beyond NF-κB activation, we tested combination of MI-2 with inhibitors of other proteins in this pathway affecting other branches of this pathway. Combination of MI-2 with the pan PI3K inhibitor BKM120, that was in our hands the most effective against a broad group of ABC-DLBCL cell lines, resulted in synergistic cell killing of OCI-Ly10 and Rc-K8 and had an additive effect in HBL-1 while it was less than additive for OCI-Ly3 and TMD8. These cell lines harbor mutations in different proteins of the pathway, which may contribute to the differences in response to the combination. Finally MI-2 strongly synergized with BH3 mimetics (most notably ABT-737) that target fundamental complementary survival pathways to BCR signaling in ABC-DLBCLs. Synergistic killing was at least partially due to induction of apoptosis, as concurrent administration of the two drugs induced increased apoptosis assessed by Caspase-7/3 activity and Annexin V+ DAPI- flow cytometry.

In summary, we identified the first specific MALT1 inhibitor drug and demonstrated a promising role for MALT1 targeted therapy as an anchor of rational combinatorial therapy against ABC-DLBCL.

Citation Format: Lorena Fontan, Chenghua Yang, Himaly Shinglot, Venkataraman Kabaleeswaran, Volpon Laurent, Michael Osborne, Elena Beltran, Monica Rosen, Rita Shaknovich, Shao N. Yang, Randy D. Gascoyne, Leandro Cerchietti, Jose A. MArtinez-Climent, J Fraser Glickman, KAtherine Borden, Hao Wu, Ari Melnick. MALT1 inhibition as an anchor for combinatorial therapy of ABC-DLBCL. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A19.