B-cell receptor (BCR) signaling plays an important role in the pathogenesis of mantle cell lymphoma (MCL), but the detailed mechanisms are not fully understood. In this study, through a genome-wide loss-of-function screen, we identify carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) as an essential factor in a subset of MCL tumors. Our signal transduction studies reveal that CEACAM1 plays a critical role in BCR activation through involvement in two dynamic processes. First, following BCR engagement, CEACAM1 co-localizes to the membrane microdomains (lipid rafts) by anchoring to the F-actin cytoskeleton through the adaptor protein filamin A. Second, CEACAM1 recruits and increases the abundance of SYK in the BCR complex leading to BCR activation. These activities of CEACAM1 require its cytoplasmic tail and the N-terminal ectodomain. Considering that previous studies have extensively characterized CEACAM1 as an ITIM-bearing inhibitory receptor, our findings regarding its activating role are both surprising and context-dependent, which may have implications for BCR-targeting therapies.

Up and downregulated proteins in Mino cells treated with 5 nM of venetoclax for 24 h, analyzed by RPPA analysis. Highlighted boxes show commonly altered proteins between Mino cells treated with venetoclax and DLBCL cell lines treated with venetoclax.

PDF - 1267KB, The established MCL cell lines, JVM-2, JVM-13, MAVER, NCEB-1, and Z-138 were treated with 20 nM CFZ for 0, 6, 12, or 24 hours and apoptosis was detected by Annexin V-binding assays. Representative histograms (left panel) and percentages of apoptotic cells (right panel) are shown.

(A) Immunoblotting of BCL-2 levels in representative MCL and DLBCL cell lines with actin as a loading control. Cell viability assays were performed using the Celltiter-Glo Luminescent Cell Viability Assay in representative GCB-DLBCL (B), non-GCB-DLBCL (C), and MCL (D) cell lines treated with increasing concentrations of venetoclax.

Supplementary Figures S1 to S3

Supplementary Table S1. Clinicopathologic characteristics of patients with de novo DLBCL with dual MYC/TP53 aberrations Supplementary Table S2. Frequency of single or dual abnormal Myc protein, p53 protein, BCL2 protein, MYC gene, and TP53 gene in the studied patients with DLBCL treated with R-CHOP Supplementary Table S3. Prognostic factors by univariate analysis and multivariate analysis in DLBCL Supplementary Table S4. Gene expression signatures identified by comparing DLBCL patients with concurrent MYC-R Mut-TP53 or Mychigh Mut-TP53 alterations (double-positive) with DLBCL patients withSupplementary Table S5. Molecular, genetic and phenotypic status of 8 DLBCL/HGBCL cell lines by targeted next-generation sequencing, fluorescence in situ hybridization (FISH), and immunohistochemistry analysis none or the alterations (double-negative) Supplementary Figure S1. Morphologic and immunophenotypic features of DLBCL with MYC rearrangement (MYC-R) and TP53 mutation (Mut-TP53) dual-alterations and highgrade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) in representative patients. Supplementary Figure S2. Prognostic impact of p53 and Myc protein overexpression as single or double abnormalities in overall DLBCL and GCB/ABC subtypes. Supplementary Figure S3. Heatmap for gene expression signatures of MYC/TP53 dual alterations. Supplementary Figure S4. Representative figures of flow cytometric analysis indicating G2/M phase-cell cycle arrest by NCB057643 treatment for 24 hours in cells with MYC-R or Myc overexpression with Wt-TP53 (OCI-LY19) or Mut-TP53 (GR and TMD8). Supplementary Figure S5. Heatmap for significantly up- or downregulated proteins after INCB057643 treatment (5µM, 24 hours) in OCI-LY19 cells. Supplementary Figure S6. A MDM2 inhibitor DS3032b shows cytotoxic effects selectively in high-grade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) and wild-type (Wt) TP53. Supplementary Figure S7. Combined DS3032b and INCB057643 treatment has no synergistic cytotoxicity in DLBCL cell lines with MYC aberrations and TP53 mutation (MutTP53). Supplementary Figure S8. INCB057643 treatment (1.25 µM) alone or in combination with ABT-199 (venetoclax, 6.25 mM) for 24 hours induced p21 expression in TMD8 cells, whereas had no effect on p53 (mutant) expression levels.

High grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH) represents an uncommon B-cell lymphoma (BCL) with aggressive clinical courses and poor prognosis. Despite revolutionary therapeutic advances in BCL, there has been limited treatment progress in HGBCL-DH, thus necessitating additional therapeutic strategies for HGBCL-DH. This study demonstrated that the BET antagonist INCB057643 synergized with the XPO1 inhibitors (selinexor and eltanexor) to decrease cell viability and increase cell apoptosis in HGBCL-DH cells with or without TP53 mutations. As anticipated, the combined treatment of INCB057643 with selinexor slowed tumor growth and reduced the tumor burden in TP53-mutated HGBCL-DH xenografts. Mechanistically, MYC functional inhibition was a potential molecular mechanism underlying the synergy of the combined INCB057643 and selinexor treatment in HGBCL-DH cells independent of TP53 mutation status. In TP53 mutated HGBCL-DH cells, inducing DNA damage and impairing the DNA damage response (DDR) were involved in the therapeutic interaction of the combined regimen. In TP53 wild-type cells, the molecular mechanism was linked with upregulation of p53 levels and activation of its targeted pathways, rather than dysregulation of the DDR. Collectively, we might provide a potential promising combination therapy regimen for the management of HGBCL-DH. Clinical evaluations are warranted to confirm this conclusion.

Background Specific B cell malignancies, including the aggressive non-germinal center B cell-like (GCB) subtype of diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), are driven by constitutive activation of the transcription factor NF-kB. Chronic signaling resulting from mutations or antigen-induced activation of the B cell receptor (BCR) pathway drives NF-kB activity in these tumors resulting in sustained proliferation and survival pathways. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) represents a key mediator of the BCR/NF-kB signal transduction pathway responsible for maintaining survival and driving proliferation of NF-kB-addicted B cell tumors by proteolytically cleaving NF-kB antagonists. Aims Pharmacological inhibition of MALT1 protease activity may provide an attractive treatment option for patients with these cancers. Further, as combination therapy is often required for the treatment of aggressive B cell malignancies, the identification of therapies that synergistically combine with MALT1 inhibitors could afford additional and promising treatment options. Methods A highly potent and orally bioavailable MALT1 protease inhibitor (ABBV-MALT1) was used to test the hypothesis that MALT1 inhibition will abrogate the proliferation of preclinical models of B cell malignancies in vitro and in vivo. Tumors treated with ABBV-MALT1 were subjected to transcriptomic and functional proteomic assays to elucidate molecular mechanisms of action and rational combination partners. Results ABBV-MALT1 potently inhibits MALT1 activity in vitro while showing high selectivity against a panel of proteases, kinases and other types of receptors and enzymes. Mechanistic studies reveal that ABBV-MALT1 effectively inhibits signal transduction of the BCR pathway and reduces NF-kB gene activation in non-GCB DLBCL cell lines resulting in cell cycle arrest and diminished viability. In vivo, oral administration of this compound demonstrates robust tumor growth inhibition in several models of B cell tumors, including non-GCB DLBCL models that are resistant to Bruton's tyrosine kinase (BTK) inhibitors. NF-kB target genes include the pro-survival family members BCL-XL and BCL2-A1, which aid in regulation of the intrinsic apoptosis pathway. As ABBV-MALT1-induced inhibition of the NF-kB pathway resulted in downregulation of these genes, we hypothesized that the associated tumor models would become increasingly dependent on the pro-survival family member BCL-2. To test this hypothesis, combination studies of ABBV-MALT1 and the selective BCL-2 inhibitor venetoclax were performed in both cell line and patient-derived xenograft models of DLBCL. Herein we show that concomitant administration of ABBV-MALT1 and venetoclax results in dramatic antitumor activity in all models tested in vivo. This efficacy also translates to primary patient CLL cells in vitro where the combination confers greater levels of apoptosis compared to either agent alone. Summary/Conclusion: ABBV-MALT1 demonstrates robust single agent anti-tumor activity in malignant B cell models that are resistant to BTK inhibitors. Moreover, combination of ABBV-MALT1 with the BCL-2 inhibitor venetoclax shows synergistic cell killing of B cell tumors in vitro and dramatic tumor regression in vivo. Together, these data indicate that MALT1 inhibition may overcome BTK inhibitor resistance and combine with venetoclax to effectively treat patients with DLBCL, CLL and other B cell malignancies. Disclosures All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Supplementary Figure Legends

Supplementary Data from Degrasyn Potentiates the Antitumor Effects of Bortezomib in Mantle Cell Lymphoma Cells In vitro and In vivo: Therapeutic Implications

Representative venetoclax-resistant (BJAB and HT) cell lines were treated with venetoclax at a 1:1 ratio drug combinations with PI3K inhibitors (idelalisib and KA2237) or an AKT inhibitor (MK-2206) in a concentration-dependent manner for 72 h, and cell viability was assessed. The highest starting concentration for each drug was 20 mM. The following are the drug combinations: 100 nM: 20 mM; 50 nM:10 mM; 25 nM:5 mM; 12.5 nM:2.5 mM; 6.25 nM:1.25 mM; 3.1 nM:0.61 mM; 1.5 nM:0.3 mM. Data from two independent experiments performed in triplicate are shown. (B) MAPK-pT202-Y204, MEK1-pS217-S221, and JNK-pT183-Y185 protein levels were plotted against the corresponding venetoclax IC50 for each cell line. Spearman's rank correlation coefficient, and p values determined of the above analysis. P values less than 0.05 indicate significant correlations.

Representative heatmap showing the up and downregulated proteins in the Mino parental (Mino-P) vs. Mino-venetoclax resistant (Mino-VR) cell lines.

Eight MCL Cell lines were cultured for 48 hours in the presence of different concentrations of CFZ. Cell growth was assessed by 3H-thymidine incorporation assay. The IC50 were determined by constructing a dose-response curve. Averages from 3 independent experiments are shown.

FOXM1 is a transcription factor that controls cell cycle regulation, cell proliferation, and differentiation. Overexpression of FOXM1 has been implicated in various cancer types. However, the activation status and func-tional significance of FOXM1 in diffuse large B cell lymphoma (DLBCL) have not been well investigated. Using pro-teomic approaches, we discovered that the protein expression levels of FOXM1 and PLK1 were positively correlated in DLBCL cell lines and primary DLBCL. Expression levels of FOXM1 and PLK1 mRNAs were also significantly higher in DLBCL than in normal human B cells and could predict poor prognosis of DLBCL, particularly in patients with germinal center B cell-like (GCB) DLBCL. Furthermore, proteomic studies defined a FOXM1-PLK1 signature that consisted of proteins upstream and downstream of that axis involved in the p38-MAPK-AKT pathway, cell cycle, and DNA damage/repair. Further studies demonstrated a mechanistic function of the FOXM1/PLK1 axis in connection with the DNA damage response pathways regulating the S/G2 checkpoint of the cell cycle. Therapeutic targeting of FOXM1/PLK1 using a FOXM1 or PLK1 inhibitor, as well as other clinically relevant small-molecule inhibitors target-ing ATR-CHK1, was highly effective in DLBCL in vitro models. These findings are instrumental for lymphoma drug discovery aiming at the FOXM1/PLK1/ATR/CHK1 axis.

High-grade B-cell lymphoma with concurrent MYC and BCL2 rearrangements (HGBL-DHL) is a rare, aggressive mature B-cell malignancy with a high likelihood of treatment failure following front-line immunochemotherapies. Patients with HGBL-DHL who develop a relapsed or refractory disease have little effective therapeutic strategies and show very poor clinical outcomes, thus calling for development of novel therapies for this specific patient population. In this study, we investigated the preclinical anti-lymphoma efficacies and potential mechanism of action of a novel treatment approach, combining the BCL2 inhibitor venetoclax with CS2164, a new orally active multitarget inhibitor, in HGBL-DHL models. This combination therapy exhibited a robust synergistic cytotoxicity against HGBL-DHL cells, evidenced by cooperatively inducing loss of cell viability and promoting cell apoptosis. Moreover, coadministration of CS2164 and venetoclax resulted in significant superior suppression of HGBL-DHL cell growth and remarkably abrogated tumor burden in a HGBL-DHL-xenografted mouse model. The synergistic lethality of CS2164 and venetoclax in HGBL-DHL cells was associated with induction of DNA damage and impairment of DNA repair ability. Of importance, the combined treatment almost abolished the expression of both BCL2 and MYC, two hallmark proteins of HGBL-DHL, and substantially blunted the activity of PI3K/AKT/mTOR signaling cascade. In addition, MCL1 and BCL-XL, two well-characterized contributors for venetoclax resistance, were significantly lessened in the presence of CS2164 and venetoclax, thus leading to the accumulation of proapoptotic proteins BAX and PUMA and then initiating the intrinsic apoptosis pathway. Taken together, these findings suggest that the regimen of CS2164 and venetoclax is highly effective to eliminate HGBL-DHL cells in the preclinical setting, warranting further clinical investigations of this regimen for the treatment of unfavorable HGBL-DHL patients.

— Nanocrystalline La 1 – x Cd x FeO 3 ( x = 0, 0.05, 0.1, 0.15, 0.2) powders with a narrow homogeneity range ( x max = 0.09 according to X-ray microanalysis and X-ray diffraction data) have been synthesized via coprecipitation followed by thermal annealing at 950°C for 1 h. The addition of Cd 2+ cations leads to a decrease in average crystallite size from 10–70 nm at x = 0 to 5–60 nm at x = 0.1 (according to transmission electron microscopy data). The synthesized nanocrystals have ferrimagnetic properties.

Single-phase nanostructured YFe1-xNixO3 (x = 0.1; 0.15; 0.2 and 0.25) perovskites were formed after annealing a mixture of precipitated hydroxides at 800 degrees C for 1 h. The results showed that at the amount of Ni substitution over 0.3, NiO and Y2O3 phases were formed in parallel with the YFeO3 phase. Moreover, as the amount of Ni substitution increased in the range of 0.1-0.5, the crystal sizes increased from 20 +/- 3 to 22 +/- 3 nm, while unit cell volumes decreased from 223.8 to 223.6 angstrom(3). The magnetic parameters (H-c, M-r, M-s) of the nanostructured Ni-doped YFeO3 perovskites were quite higher than those of pure YFeO3, as well as of YFeO3 doped Ca or Co. As the amount of Ni substitution is in the range of 0.1-0.25, H-c decreased from 1332.6 to 887.9 Oe, while M-s increased from 0.67 to 1.18 emu/g and M-r increased from 1.8.10(-1) to 3.2.10(-1) emu/g. (C) 2020 Elsevier B.V. All rights reserved.

This paper reports the study of optical and magnetic properties of HoFeO3 nanocrystals prepared by a simple co-precipitation method using ethanol. Single-phase HoFeO3 was formed after calcination of the precipitate at 650, 750 and 850 degrees C during 1 h. The XRD and TEM results showed that the average crystallite sizes of all HoFeO3 samples were in the range of 25-30 nm. The UV-Vis spectra showed strong UV and Vis absorption with a small optical energy gap (E-g = 1.8019 eV). The soft ferromagnetic behavior of HoFeO3 nanoparticles with magnetization similar to 0.7-0.8 emu/g, coercivity similar to 8-23 Oe, remanent magnetization less than 0.005 emu/g. (C) 2020 Elsevier B.V. All rights reserved.

In this study, a nanostructured NdFeO3 material was synthesized via a simple process of the hydrolysis of Nd (III) and Fe (III) cations in hot water with 5% NaOH as a precipitating agent. According to the results of the thermal behaviors of each hydroxide, either containing Fe (III) or Nd (III), the perovskite type of neodymium orthoferrite NdFeO3 was simply synthesized by annealing a mixture of Fe (III) and Nd (III) hydroxides at 750 °C. The nanostructured NdFeO3 was obtained in spherical granules with diameters of around 30 nm. The magnetic properties of the material were a coercive force (Hc) of 136.76 Oe, a remanent magnetization (Mr) of 0.68 emu·g–1, and a saturation magnetization (Ms) of 0.79 emu·g–1.

Abstract Diffuse large B-cell lymphoma (DLBCL) is the major type of aggressive B-cell lymphoma. High-grade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) represents a distinct entity with dismal prognosis after standard immunochemotherapy in the current WHO lymphoma classification. However, whether TP53 mutation synergizes with MYC abnormalities (MYC rearrangement and/or Myc protein overexpression) contributing to HGBCL-like biology and prognosis is not well investigated. In this study, patients with DLBCL with MYC/TP53 abnormalities demonstrated poor clinical outcome, high-grade morphology, and distinct gene expression signatures. To identify more effective therapies for this distinctive DLBCL subset, novel MYC/TP53/BCL-2–targeted agents were investigated in DLBCL cells with MYC/TP53 dual alterations or HGBCL-MYC/BCL2-DH. A BET inhibitor INCB057643 effectively inhibited cell viability and induced apoptosis in DLBCL/HGBCL cells regardless of MYC/BCL2/TP53 status. Combining INCB057643 with a MDM2-p53 inhibitor DS3032b significantly enhanced the cytotoxic effects in HGBCL-DH without TP53 mutation, while combining with the BCL-2 inhibitor venetoclax displayed potent therapeutic synergy in DLBCL/HGBCL cells with and without concurrent TP53 mutation. Reverse-phase protein arrays revealed the synergistic molecular actions by INCB057643, DS3032b and venetoclax to induce cell-cycle arrest and apoptosis and to inhibit AKT/MEK/ERK/mTOR pathways, as well as potential drug resistance mechanisms mediated by upregulation of Mcl-1 and RAS/RAF/MEK/ERK pathways. In summary, these findings support subclassification of DLBCL/HGBCL with dual MYC/TP53 alterations, which demonstrates distinct pathobiologic features and dismal survival with standard therapy, therefore requiring additional targeted therapies. Implications: The clinical and pharmacologic studies suggest recognizing DLBCL with concomitant TP53 mutation and MYC abnormalities as a distinctive entity necessary for precision oncology practice. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/19/2/249/F1.large.jpg.