ALBERT

Description: 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

Description: Background: The prognosis of multiple myeloma (MM) has improved significantly over the last 20 years due to the introduction of autologous stem cell transplantation and novel drugs. The standard regimen for conditioning in MM is melphalan 200 mg/m2, since a study published in 2002 (Moreau et al.) showed a survival advantage of melphalan (MEL) only over melphalan- total body irradiation (MEL-TBI) conditioning. Ionizing radiation is an independent treatment modality and has activity in MM. We decided to reevaluate the outcome of MEL-TBI conditioning in the age of novel drugs. Patients and Methods: In a retrospective chart review, we identified 50 patients with MM who underwent autologous hematopoietic cell transplantation at Tulane University Medical Center and were conditioned with MEL-TBI between December 1995 and March 2012. Stem cells were collected following a stimulation by 10 µg/ kg filgrastim for 4 days. In case of insufficient collection, plerixafor was administered. Between 1995 and 2003 cyclophosphamide priming was used in most cases. Patients received melphalan 140 mg/m2 as a single dose given intravenously on day -5. TBI was administered in fractionated doses of 150 cGy between days -4 and -1 (total dose 12 Gy). Peripherally harvested stem cells were infused on day 0. Standard supportive measures were followed. Log-rank test and Kaplan-Meier curves were used to calculate overall survival (OS) and progression-free survival. Significance levels were standard (p