Mapping MALT1 Signaling Connectivity Unveils Novel B-Cell Feedback Mechanisms Directing Assembly of Potent Anti-Lymphoma Regimens
Authors: Fontan Lorena , Durant Matthew , Goldstein Rebecca , Teater Matthew R , Casalena Gabriella , Xia Min , Wilson Jimmy , Us Ilkay , Inghirami Giorgio , Melnick Ari 
Published: February 2020

MALT1 inhibition is a promising strategy against B-cell receptor (BCR)-dependent lymphomas including ABC DLBCL, CLL and MCL. MALT1 is downstream of the most frequently mutated genes in the BCR and Toll-like receptor (TLR) pathways. MALT1 inhibitors are active in Ibrutinib-resistant BTK and PLCγ2 mutant CLL or CARD11 mutant ABC DLBCL. Therefore, MALT1 inhibitors, which recently began first in man clinical testing, have the potential to cover a larger patient population than drugs against more upstream targets in the BCR pathway including BTK inhibitors. However, activity of inhibitors targeting signaling mediators can be limited by feedback mechanisms counteracting and/or bypassing the need for a specific pathway. MALT1 is central to NF-κB activation downstream of the BCR. Therefore, activation of alternative pathways leading to full or partial activation of the BCR program or other pro-survival pathways might enable cell survival and set off MALT1i resistance.
In order to map the landscape of resistance/sensitivity to MALT1 inhibition in ABC DLBCL and assist design of combinatorial regimens, we carried out a loss-of-function screen in HBL-1 (MALT1i sensitive) with or without MI-2 (small molecule irreversible inhibitor of MALT1) to identify genes capable of modulating response to MALT1 inhibition. Our analyses showed that loss of BCR and PI3K activators enhanced sensitivity, while loss of negative regulators of these pathways promoted MALT1i resistance. These findings were validated by knockdown of individual genes with two independent hairpins against activators CD79B, CARD11, BTK or the negative regulator TNFAIP3.
Next, we carried out a combinatorial drug screen anchored in MALT1 inhibition by MI-2 or C3 (irreversible substrate-mimetic MALT1 inhibitor) and focused on inhibitors against signaling hubs in the BCR/PI3K pathways in 4 MALT1i sensitive cell lines. This combinatorial screen confirmed that concurrent inhibition of MALT1 and other BCR and PI3K pathways' proteins is additive (0.9&gt;CI (combination index) &lt;1.1) or synergistic (CI&lt;0.9). MALT1i combinations with PI3K and MTORC1 inhibitors were the most highly synergistic, mean CI&lt;0.5 for 2 MALT1 inhibitors and 4 cell lines, and were further pursued.
In depth analysis of proliferation and cell death by CFSE dilution and Annexin V staining revealed that both MALT1/PI3K-i and MALT1/MTORC1-i combinations significantly enhanced growth inhibition and apoptosis in TMD8 and HBL-1 compared to individual agents. Results with MI-2 and C3 were comparable. Short exposure to MI-2 or C3 increased MTORC1 activity as assessed by S6K-Thr389 and S6-Ser235/6 phosphorylation in TMD8 and HBL-1 indicating that MALT1 protease activity modulates MTORC1 activation. MTOR activation is tumorigenic and can mediate chemotherapy resistance. Increased p-S6 following MALT1 inhibition (FC=1.4-2) was blocked by Idelalisib (PI3Ki). However, only Rapamycin, an MTORC1 inhibitor, reduced p-S6 levels relative to vehicle, FC=-5 alone or in combination.
In vivo, MALT1/PI3K-i (MI-2/Idelalisib) significantly delayed tumor progression compared to single treatments (p&lt;0.01). In contrast, MALT1/MTORC1-i (MI-2/Rapamycin) promoted tumor regression and significantly improved survival of xenografted mice (median survival 37 days vs 18.5 or 29 days for MI-2 or Rapamycin respectively, p&lt;0.001). Tumors from MI-2/Idelalisib treated mice showed 15-fold increase in p-S6 at 21 days. Short exposure to MI-2 in vivo inhibited MALT1 activity over its targets BCL10 and Roquin-1 and increased MTORC1 activity over p-S6K-Thr389 (FC=1.5-2) and p-S6-Ser235/6 (FC=2-3) in ABC DLBCL xenografts. Rapamycin, but not Idelalisib, effectively blocked p-S6 (p&lt;0.001).
MALT1/MTORC1-i regimens were also highly synergistic in 2 patient-derived xenografted (PDX) ABC DLBCL ex vivo. PDXs were cultured in gelatin/silicate nanoparticle hydrogel 3-D organoids and co-cultured with CD40L expressing cells. Compound pairs were assayed for synergy using 4x4 matrices and growth inhibition evaluated by flow cytometry. Synergy ZIP δ-score ranged 7-14 in 2 specimens for 2 MALT1i.
Combined, these results suggest that: 1) MTORC1 activation constitutes a survival feedback mechanism activated after MALT1i treatment that might be leveraged by tumoral cells to evade MALT1 inhibition and, 2) that simultaneous targeting of MTORC1 could improve response and prevent resistance to MALT1 inhibitors.

Disclosures
Melnick: Constellation: Consultancy; Janssen: Research Funding; Epizyme: Consultancy.