Targeting MALT1 with the Small Molecule Inhibitor MI2 Induces a Caspase-Dependent Apoptosis and Inhibits the NF-κb Pathway in Chronic Lymphocytic Leukemia Primary Cells
Authors: Tanios Georges , Lobelle-Rich Patricia , Vasudevamurthy Ashwin , Atkinson Thomas S. , Baghian Ali , Liu Delong , Safah Hana , Flemington Erik K. , Saba Nakhle S. 
Published: October 2019

Abstract
Targeting the B-cell receptor (BCR) pathway in chronic lymphocytic leukemia (CLL) with the Bruton Tyrosine Kinase (BTK) inhibitor Ibrutinib has transformed the treatment paradigm of this disease. Ibrutinib is currently indicated for treatment of CLL regardless of the line of therapy. However, ibrutinib is not curative, and relapses secondary to C481S mutation in BTK and gain of function mutations in PLCγ2 (R665W and L845F) are being reported.
The protease mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) is the active component of the CARD11-BCL10-MALT1 (CBM) signaling complex. CBM mediates NF-κB activation downstream of BTK and PLCγ2 within the BCR pathway, which makes of MALT1 an ideal therapeutic target in ibrutinib-resistant clones. Notably, the chemical compound MI2 (C19H17Cl3N4O3) binds to- and suppresses the protease activity of MALT1, and was found to be active against activated B-cell-like diffuse large B-cell lymphoma. However, the role of MALT1 inhibition in CLL has not been investigated.
We studied the efficacy of MI2 against a cohort of CLL samples (N=21), and explored its mechanism of action. PBMCs from patients with CLL were tested against serial dilutions of MI2 (0.125-8μM) for 48 h in 96-well plates, and MTS assay was performed to quantify cell viability. We observed a dose-dependent cytotoxicity in all samples, with an inhibitory concentration at 25% (IC25) < 2μM observed in the majority of the samples. MI2 cytotoxicity was independent of the IGHV mutational status, CD38 expression, cytogenetics, previous therapy status, and MALT1 protein level (immunoblot on purified CLL cells). To study the drug apoptosis and drug selectivity, we examined apoptotic induction in both CLL cells and T-cells of the same patients. PBMCs from five patients with CLL were treated with MI2 for 48 h and compared to untreated control. At the end of the incubation, cells were stained with CD5, CD19, and Annexin-V, then analyzed using flow cytometry. We found that MI2 induced a dose-dependent apoptosis in CLL cells (defined as CD5+/CD19+) in all samples, with only mild toxicity to their corresponding T-cells (T-cells represent a fraction of each patient's PBMCs defined as CD5+/CD19-). This minimal toxicity to T-cells was not statistically significant when compared to untreated control T-cells, and may reflect a T-cell receptor signaling-independence of circulating T-cells, which are mostly composed of terminally differentiated effectors. To determine whether the induced apoptosis is caspase-dependent, we treated PBMCs of CLL patients (N=5) with MI2 (0.5-4 μM) in the presence or absence of 100μM of the pan-caspase inhibitor z-VAD-fmk for 48 h. We found that z-VAD-fmk completely prevented the induction of apoptosis in CD19+/CD5+ CLL cells as determined by Annexin-V staining (flow cytometry), and decreased MI2-induced PARP cleavage (Immunoblot).
To determine the effect of the microenvironment on MI2 toxicity, we cultured primary CLL cells with and without MI2 (0.5-4µM) for 48 h, in the presence or absence of Nurse-Like Cells (NLC) (N=5) or anti-IgM (N=4). NLCs and anti-IgM cross-linking of BCR are in-vitromodels for the protective effect of the microenvironment. Co-culture with NLCs or with anti-IgM improved the viability of the untreated primary CLL samples. Similar to ibrutinib, MI2 continues to be effective against CLL cells in the presence of NLCs or anti-IgM, but higher concentrations are required to achieve the desired cytotoxicity against CLL cells. There was no cytotoxic effect on NLCs.
To investigate the effect of MI2 on tumor biology, 1 μg of total RNA from purified CLL cells (treated for 8 h with MI2, N=3; and untreated control, N=3) was subjected to RNA sequencing. Out of 56,650 tested genes, there were 438 genes whose expression changed ≥2-fold at P<0.05 (312 down- and 126 up-regulated), several genes are known NF-κB targets. The observer-independent method, Gene Set Enrichment Analysis (GSEA), clearly identified the canonical NF-κB among of the top affected pathways.
Further studies aim to verify the RNA sequencing data at the protein level, to determine MI2 efficacy against ibrutinib-resistant clones in vitro, and against CLL growth in mice in vivo.
Supported by Hope Foundation SWOG Early Exploration and Development (SEED) Award. We thank the Louisiana Cancer Research Center (LCRC) biospecimen core and our patients for the donation of samples to make this research possible.

Disclosures
No relevant conflicts of interest to declare.