ALBERT

Description: White blood cells from 22 patients with leukemia and lymphoma were studied for the presence of terminal deoxynucleotidyl transferase with a peroxidase-antiperoxidase method. The enzyme was detected in leukemic cells of 5 patients with acute lymphoblastic leukemia and 1 patient with chronic myelogenous leukemia, whereas 16 patients with different forms of leukemia or lymphoma were negative for this enzyme. Comparative studies using a biochemical and an indirect immunofluorescence assay revealed complete concordance between these three methods.

Description: White blood cells from 22 patients with leukemia and lymphoma were studied for the presence of terminal deoxynucleotidyl transferase with a peroxidase-antiperoxidase method. The enzyme was detected in leukemic cells of 5 patients with acute lymphoblastic leukemia and 1 patient with chronic myelogenous leukemia, whereas 16 patients with different forms of leukemia or lymphoma were negative for this enzyme. Comparative studies using a biochemical and an indirect immunofluorescence assay revealed complete concordance between these three methods.

Description: MALT1 is a protease and scaffold protein involved in signal transduction to NF-κB downstream of several receptors including B-cell (BCR) and T-cell receptors (TCR). MALT1 is aberrantly activated in ABC DLBCL by mutations in upstream genes in the BCR and TLR pathways (CD79A/B, CARD11, MYD88) and is critical for proliferation and survival. Recent studies by our lab, and others, identified inhibitors of MALT1 protease activity that revealed MALT1 is therapeutically targetable in ABC DLBCL. MALT1 is also essential for CLL, MCL and certain solid tumors (most notably lung cancer and glioblastoma). A first-in-man clinical trial recently started to evaluate MALT1 protease inhibition in B-cell non-Hodgkin's lymphomas. However, chronic inactivation of MALT1 protease activity suppressed T regulatory cells in vivo in protease dead murine models causing fast progressing autoimmune disease and death. Loss of MALT1, on the other hand, has also potent anti-tumoral effects but does not lead to autoimmunity in murine models. These findings prompted us to study alternative MALT1 targeting therapeutic approaches aimed to target its scaffolding activity. We have developed a series of proteolytic targeting chimera (PROTAC) compounds against MALT1. PROTACs are bifunctional compounds that induce selective proteolysis by targeting proteins of interest to E3 ligases for directed proteosomal degradation. Our MALT1 PROTACs are based on an allosteric MALT1 inhibitor that binds reversibly to MALT1 and fused to a Cereblon (CRBN) binding moiety, with the intent of bringing the CRBN E3 ligase complex in close proximity of MALT1 and promoting its ubiquitination and proteasomal degradation. We performed structure-activity relationship analysis and studied: 1) three linker attachment points in the MALT1 binding moiety and evaluated their MALT1 enzymatic inhibitory activity and binding to MALT1; 2) the effect of distinct linker length and polarity in MALT1 degradation and, 3) compared the effect of Lenalidomide, Pomalidomide and CC-220, which have increasing CRBN affinities, as alternative CRBN-binding moieties. Six out of eighteen compounds presented higher than 50% MALT1 degradation at 1 μM compared to vehicle treated cells in a MALT1-dependent cell line, OCI-Ly3. The parental allosteric compound, on the other hand, did not affect MALT1 levels compared to vehicle treated cells and was used as a negative control for MALT1 degradation. Compounds that actively degraded MALT1 over 50% preserved selective killing of ABC DLBCL over GCB DLBCL, same as the parental MALT1 inhibitor. GI50 of active compounds in OCI-Ly3 was 2-6 μM while it was greater than 20 μM for the MALT1-independent cell line OCI-Ly1. We chose two of our most effective and selective compounds to validate MALT1 PROTACs mechanism of action. Unlike their parental MALT1 targeting allosteric compound, MALT1 PROTACs effectively degraded MALT1 in a CRBN-dependent manner as shown in a 293T-CRBN knockout cell line or in OCI-Ly3 cells by treatment with 1 μM MLN4924. MLN4924 inhibits NEDD8-activating enzyme which is essential for the CRBN complex to function. Notably, MALT1 PROTACs degraded MALT1 in OCI-Ly1 cells (FC=-2.5) and Raji cells (FC=-1.7), where MALT1 is inactive. MALT1 degradation by PROTACs was not affected by activation in Raji cells, since PMA/ionomycin treatment did not alter the effect of MALT1 PROTACs on MALT1 levels. Therefore, MALT1 PROTACs can degrade MALT1 independent of its activation state. Moreover, unlike MALT1 protease inhibitors, MALT1 PROTACs potently suppress NF-κB activation, which is dependent on MALT1 scaffolding activity, as assessed by WB of phopho and total IκB in ABC DLBCL cell lines. Our data shows that MALT1 PROTACs could be excellent agents for the treatment of ABC DLBCL and other lymphomas, providing an alternative to enzymatic targeting that might prove useful to avoid autoimmunity or overcome resistance mechanisms. Disclosures Gray: Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Melnick:Constellation: Consultancy; Janssen: Research Funding; Epizyme: Consultancy.

Description: Recent studies have identified small molecule inhibitors of the paracaspase activity of MALT1, a protease and scaffolding protein involved in the B-cell receptor (BCR) signaling pathway, that are effective killing lymphomas in vitro and in vivo in xenograft models of Activated B-cell like Diffuse Large B-cell Lymphoma (ABC-DLBCL). ABC-DLBCL is characterized by constitutive NF-κB activity. This activation has been attributed to mutations in various protein components of the B-cell receptor (BCR) as well as Toll-like receptor (TLR) pathways. However, not all ABC-DLBCL cell lines and primary patient samples were equally sensitive to MALT1 inhibitors in vitro. In order to discover genetic modifiers of response to MALT1 inhibition we used an shRNA library screening approach. MALT1 inhibition sensitive cell line HBL-1 was infected with DECIPHER barcoded shRNA library Module 1 and cells were treated with vehicle or 300 nM of MALT1 inhibitor MI-2 for 22 days. At this time cells were harvested and genomic DNA extracted. PCR was used to amplify barcodes and gel purified bands were extracted and sequenced. Cellecta's Deconvoluter software was used to quantify the number of reads per shRNA, reads were normalized to total number of reads and fold change between vehicle and MI-2 treated cells was calculated. Among the top positively and negatively enriched hairpins, we found a significant number of genes involved in the BCR pathway including: positively regulated shRNAs against TNFAIP3 and FOXO1 and negatively regulated hairpins against BTK, CD79B and PI3K genes PIK3C2A and PIK3C2D. Interestingly, TNFAIP3 and FOXO1 are negative regulators of the BCR pathway while BTK, CD79B and PI3K genes are positive regulators of this pathway. In order to validate these results and given the abundance of inhibitors of different proteins in the BCR pathway, we run a focused combination screen using MALT1 inhibitor MI-2 and inhibitors against other proteins in the pathway in 4 MALT1 sensitive cell lines. Combinations with PI3K inhibitors were most synergistic (combination index (CI) ranging 0.12-0.67), while BTK and PKC inhibitors showed an additive effect (CI ranging 0.7-0.9). These results were confirmed using a second MALT1 inhibitor, mepazine. In order to characterize the molecular mechanism by which MALT1 inhibition cooperates with PI3K, we focused on the FDA approved drug Idelalisib. In vitro treatment of cells with MI-2 and Idelalisib showed that effect on cell growth was a combination of decreased proliferation and increased apoptosis. Moreover, we found a decrease in AKT phosphorylation followed by a decrease in FOXO1 T24 phosphorylation and an accumulation of FOXO1 protein. This result correlates with our finding that FOXO1 knockdown favors MALT1 inhibition resistance. In vivo treatment of TMD8 xenografts with a combination of MI-2 and Idelalisib showed a stronger effect than either drug used as a single agent or vehicle, confirming the increased efficacy of the combination over either drug alone. In summary, we have used an shRNA library screening in order to determine which proteins and pathways cooperate with MALT1 inhibition to kill ABC-DLBCL and to evaluate combinatorial treatments in an unbiased manner. This same approach has pointed out TNFAIP3 and FOXO1 as possible biomarkers of response. This is especially interesting since these two proteins are mutated in a proportion of ABC-DLBCL patients and could affect response to treatment not only against MALT1 inhibitors but potentially any BCR targeted therapy. Disclosures Melnick: Janssen: Research Funding.

Description: 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〉CI (combination index)