OCI-Ly10 Cells Research Articles

Ars2-containing bispecific, Fab- and IgG1-format BAR-bodies to target DLBCL cells.

Despite recent advances in the therapy of diffuse large B-cell lymphoma, not otherwise specified (DLBCL), around 30% of patients develop refractory disease or relapse after first-line treatment. Recently, Ars2 was reported as the auto-antigenic target of the B-cell receptor (BCR) in approximately 25% of activated B-cell DLBCL cases. Ars2 could be used to specifically target B cells expressing Ars2-reactive BCRs. However, the optimal therapeutic format to integrate Ars2 into has yet to be determined. To mimic therapeutic antibody formats, Ars2-containing bispecific and IgG1-like constructs (BCR antigens for reverse [BAR]-bodies) were developed. Two bispecific BAR-bodies connecting single-chain antibodies against CD16 or CD3 to the BCR-binding epitope of Ars2 were constructed. Both constructs showed strong binding to U2932 cells and induced effector cell-dependent and selective cytotoxicity against U2932 cells of up to 44% at concentrations of 20μg/ml. Additionally, IgG1-format Ars2 BAR-bodies were constructed by replacing the variable heavy- and light-chain regions of a full-length antibody with the Ars2 epitope. IgG1-format Ars2 BAR-bodies also bound selectively to U2932 and OCI-Ly3 cells and induced selective cytotoxicity of up to 60% at 10μg/ml. In conclusion, Ars2-containing bispecific and IgG1-format BAR-bodies both are new therapeutic formats to target DLBCL cells.

Development of a Robust BH3 Profiling Toolkit for Precision Medicine in Hematologic Malignancies

INTRODUCTION. BCL2 homology 3 (BH3) mimetics represent a novel class of pro-apoptotic anti Hematologic malignancies (HM) drugs directly targeting apoptosis, even in chemoresistant cells. Each disease is heterogenous which makes it hard to provide standardized treatment for each patient and highlight the need for new diagnosis tools to deliver precision medicine. In the other hand, a technic called BH3 profiling has been developed, allowing to measure the apoptosis priming of the cells as well as their dependencies on specific anti-apoptotic proteins. Using specific BH3 peptides, this assay measures the percentage of cytochrome c release for each anti-apoptotic protein, mainly Bcl2, Mcl1 and Bcl-xl. BH3 toolkit, a variation of this technic, was developed using BH3 mimetics instead of BH3 peptides. Those small molecules mimic the effect of BH3 sensitizer protein by competing for anti-apoptotic protein catalytic pocket. Using these drugs allows to avoid cell permeabilization and thus reduce variability in the assay. A dynamic BH3 toolkit is also possible, measuring early drug-induced changes in net proapoptotic signaling, to identify modification of antiapoptotic dependencies. The aim of the present work is to develop a standardize and reproducible BH3 toolkit (baseline and dynamic), with internal control, to allow an absolute measurement of specific anti-apoptotic dependencies in HM cell lines or primary tumor cells, as well as identifying optimal therapeutic combination. MATERIAL AND METHODS. We used different HM cell lines as well as primary cells from patients with CLL. Cells were seeded right after thawing at the concentration of 200,000 cells/mL in a 96 well plate, using a robotic liquid handler. Primary cells were cocultured with stromal cells (Resto-6 cell line). Cells are then incubated for 20h in a 5% CO2 and 37°C incubator. Next, we treated them with BH3 mimetics (or vehicle): Venetoclax (VEN, Bcl2 inhibitor), Navitoclax (Bcl2 and Bcl-xl inhibitor), AZD-5991 (AZD, Mcl1 inhibitor), A-1155463 (Bcl-xl inhibitor) for 4h. Then, cells were stained using Annexin V and 7AAD and analyzed by flow cytometry. Our gating strategy focuses on live cells to measure annexin V flipflop as an apoptotic marker. This assay is paired with a cell viability measurement at 24h, using CellTiterGlo assay. For dynamic BH3 toolkit, the influence of the tested drug on antiapoptotic dependencies is measured by adding this drug (or vehicle) at the same time as cells of interest. RESULTS. First, we wanted to optimize the protocol on cell lines with known anti-apoptotic dependencies. We used OCI-Ly1 and LP1 cell lines which are known for their exclusive Bcl2 and Mcl1 dependencies, respectively. As expected, OCI-Ly1 cells showed a significant increase in annexin V staining (reaching 48.2% (P=0.0003) at maximum concentration) upon VEN treatment, whereas it does not change significantly with Mcl1 or Bcl-xl inhibitors (Figure A). Likewise, we detected an increase of 15.35% (P=0.005) in Annexin V staining only upon AZD in LP1 cells, but no significant modification with other inhibitors. Consistent with these data, we saw a significant and specific decrease in cell viability (CTG) with VEN and AZD on OCI-Ly1 and LP1 respectively (data no shown). These 2 cell lines are used as internal controls for the other analyses. Next, we optimized the assay on CLL primary cells. Figure A shows that VEN induces an increase in Annexin V staining by 62.7 % (P=0.0003), demonstrating a significant Bcl2 dependency. Finally, we optimized the dynamic part of the assay (Figure B). We used ironomycin, a lysosomal iron-targeting small molecule that has been studied by our team in HM (Devin et al. 2022). We found that ironomycin increased significantly Bcl2 dependency in OCI-Ly1 cells by 8.55% (P=0.02) with VEN. Interestingly, we observed the induction of a MCL1 dependency in those cells, with an annexin V staining increase by 8.21% (P=0.02). CONCLUSION. We were therefore able to measure the baseline dependencies using a BH3 toolkit that avoid cell permeabilization. We were also able to measure changes in dependencies induced by an innovative therapy. We believe that our results show the robustness of the BH3 toolkit and its ability to detect potential new therapeutic strategies in HM cell lines and primary cells. Detailed results will be presented at the meeting, as well as data on primary lymphoma and myeloma cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Investigations of Redox Resistance in Aggressive Lymphomas with Cholesterol Modulating Lipid Nanoparticles

Introduction: High grade B cell lymphoma (HGBCL), characterized by C-MYC and BCL-2 rearrangements, are particularly aggressive with poor response to standard therapy and decreased overall survival (OS). We previously demonstrated that B-cell lymphomas, such as germinal center (GC) diffuse large B-cell lymphomas (DLBCL), are addicted to cholesterol uptake and that bioinspired gold core lipid nanoparticles (Au-LNPs) antagonize lipid uptake through the cell surface receptor called scavenger receptor type B-1 (SR-B1) and lead to cholesterol depletion in several lymphoma cell lines (Rink et al JBC 2020). Au-LNP treatment leads to lymphoma cell death by ferroptosis, through reduction of glutathione peroxidase (GPX4) and subsequent accumulation of oxidized lipids. The antioxidant Nrf2 pathway is known to resist oxidative stress and high levels of Nrf2 proteins predict poor response to chemotherapy in hematologic malignancies. We therefore hypothesized that 1) HGBCLs would be more resistant to Au-LNP and 2) antioxidant pathways might mediate that resistance. Methods: The GC DLBCL cell line (SUDHL-4) cells and HGBCL cell lines (RC, OCI-ly1, OCI-ly8 and OCI-ly18) were used. Cells were treated with PBS (control) or Au-LNPs for 72h. Au-LNPs were synthesized as previously described (Rink JBC 2022). Cell viability was measured by MTS assay. Total RNA in RC and SUDHL-4 cells were extracted and used to construct cDNA libraries for RNA sequencing (RNAseq) by HiSeq 4000 with single-end 50bp reads and 20-25 million reads per sample in the NUSeq Core of Northwestern University. Metascape was used for functional analysis of identified differentially expressed genes (DEGs) and DEGs related to Nrf2 survival signaling, NQO1, FTH1, FTL, G6PD, TXNRD, NFE2L2, TXN, P4HB, and MGAT3 were confirmed in control and Au-LNP treated SUDHL-4, RC, OCI-ly1, OCI-ly8 and OCI-ly18 cells by RT-qPCR and Western blot analysis. Results: HGBCL cells (RC, OCI-ly1, OCI-ly8 and OCI-ly18) were more resistant to treatment with Au-LNPs (IC50 = 31.42 nM, 27.22 nM, 58.24 nM, 26.06 nM, respectively) compared with SUDHL-4 (IC50 8.14 nM). RNAseq analysis identified 320 upregulated and 162 downregulated genes in SUDHL-4 cells and 346 upregulated and 182 downregulated genes in RC cells after treatment with Au-LNPs. Of these DEGs, 62 upregulated and 8 downregulated DEGs appeared in both cell lines after treatment. The top 5 upregulated common genes include ABHD4, DUSP5, JUN, RGS1 and SQSTM1. The top 5 downregulated common genes include ABCG1, DDN, GPX1, GPX4 and IKZF1. There were 284 upregulated and 174 downregulated DEGs only identified in Au-LNP treated RC cells. Among 284 upregulated DEGs the top 2 enriched clusters were Nrf2 survival signaling and nuclear receptors meta-pathways. Au-LNP treatment in RC cells showed log2 fold increase of NQO1 (4.6), FTH1 (1.28), FTL (2.1), and TXNRD1 (1.78), genes in the Nrf2 survival signaling pathway. In SUDHL-4 cells, only NQO1 and FTL1 showed significant differential expression with log2 fold increase of 0.7 and 0.45 after Au-LNP treatment. To confirm RNAseq results, we measured the expression of Nrf2 survival signaling genes, NQO1, FTH1, FTL, G6PD, TXNRD, NFE2L2, TXN, P4HB, and MGAT3 with or without Au-LNP in SUDHL4, RC, OCI-ly1, OCI-ly8, and OCI-ly18 cells by RT-qPCR. In SUDHL4 cells, compared with PBS control, Au-LNP treatment led to mild increase in expression of NQO1 (4.2 ± 0.6 fold). By contrast, in HGBCL cells, Au-LNPs increased NQO1 expression 19.9 ± 0.2 fold for RC cells, 9.0 ± 0.3 fold for OCI-ly1 cells and 36.5 ± 2.3 fold for OCI-ly8 cells compared with PBS control. Higher NQO1 and FTH1 protein expression in treated RC cells was also confirmed by western blot analysis. These data suggest that 1) HGBCL cell lines RC, OCI-ly1, OCI-ly8 and OCI-ly18 are more resistant to cell death by cholesterol depletion and that 2) this phenomenon is mediated by upregulation of genes in the Nrf2 pathway and NQO1, in particular. Conclusion: The redox related Nrf2 survival signaling pathway, especially the NQO1 gene, is upregulated after treatment with Au-LNPs. This was observed to a greater extent in the more resistant HGBCL cells (RC, OCI-ly1, OCI-ly8 and OCI-ly18) than the SUDHL4 cells. Strategies to target the Nrf2 pathway may reverse resistance to cholesterol depletion in lymphoma.

Inhibition of PRMT5 Increases Sensitivity to BH3 Mimetics in Aggressive B Cell Malignancies

Introduction: Protein Arginine Methyltransferase 5 (PRMT5) is a type II methyltransferase that catalyzes symmetric dimethylation of arginine. PRMT5 is highly expressed in several types of aggressive B cell malignancies, and increased PRMT5 expression has been associated with poor disease prognosis. Several small molecule inhibitors targeting PRMT5 have been developed and showed promising anti-tumor effect in pre-clinical studies including in mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL). JNJ-64619178, a potent and specific PRMT5 inhibitor, exhibited broad antiproliferative activity in cancer cell lines from multiple cancer types and cell lines derived xenograft mouse model (Brehmer et al., 2021). In a phase 1 clinical trial of JNJ-64619178 (NCT03573310), patients with advanced solid tumors and non-Hodgkin lymphoma demonstrated manageable toxicity and preliminary evidence of antitumor activity at selected dose levels (Villar et al., 2020). In addition, in patients with lower-risk myelodysplastic syndromes, JNJ-64619178 had manageable hematologic toxicity at the selected expansion dose, but did not have significant clinical activity (Haque et al., 2021). Inhibition of PRMT5 alone could induce cell cycle arrest instead of apoptosis depending on the types of cancer (Zhu et al., 2019), therefore we hypothesized that the combination of PRMT5i and pro-apoptotic BH3 mimetics therapies could be synergistic in aggressive B cell malignancies. Methods: We used dynamic BH3 profiling (DBP) to measure the net pro-apoptotic signaling induced by ex vivo drug treatments (as per Montero et al., 2015). Other standard techniques included shRNA mediated gene knockdown, Western blot, cell viability assays including PI/Annexin V and CellTiter-Glo, and reactive oxygen species (ROS) measurement by flow cytometry with dihydroethidium (DHE) staining. The DLBCL cell lines (HBL1, TMD8, RI-1, OCI-Ly1, Karpas 422, SUDHL4, Toledo), double hit lymphoma (DHL) patient-derived xenograft (PDX) cell lines (DW19), mantle cell lymphoma (MCL) cell lines (Mino, Jeko-1), T cell acute leukemia (T-ALL) cell line (Jurkat) and Burkitt lymphoma (BL) cell line (Raji) were used to investigate the in vitro anti-cancer activity of JNJ-64619178 (Janssen Pharmaceuticals) and BH3 mimetics (venetoclax [BCL2i], S63845 [MCL-1i] and A1331852 [BCL-xLi], Selleckchem). SynergyFinder was used to calculate drug synergism. Results: In all 7 DLBCL cell lines and the DHL-PDX cell line, we found synergistic induction of apoptosis with JNJ-64619178 plus venetoclax (synergy score, 2.82~51.42). The lowest level of synergy was observed in OCI-Ly1 cells, which are intrinsically highly sensitive to venetoclax monotherapy. The combination of JNJ-64619178 with S63845 synergistically induced apoptosis in 5 out of 7 cell lines (synergy score, -1.91~35.64). While the combination of JNJ-64619178 with A1331852 synergistically induced apoptosis in all 7 cell lines (synergy score 1.88~46.73), 4 of them had weak synergy score (1.88~9.4). To confirm the effects we observed with pharmacologic inhibition of PRMT5, we performed shRNA mediated knockdown of PRMT5, which similarly revealed increased sensitivity to venetoclax. In MCL cell lines, combining JNJ-64619178 with venetoclax or A1331852 was more synergistic in inducing cell death than combining with S63845. The T-ALL Jurkat cell line was resistant to JNJ-64619178 and single BH3 mimetic-induced apoptosis. Combining JNJ-64619178 with A1331852 but not venetoclax or S63845 strongly induced apoptosis. The Burkitt cell line Raji was resistant to JNJ-64619178, venetoclax and A1331852, but was sensitive to S63845. The addition of JNJ-64619178 and a single BH3 mimetic in these Raji cells had only a minor synergistic effect. The synergy scores are summarized in Figure 1. Using DBP, JNJ-64619178 was found to increase overall apoptotic priming of DLBCL and MCL cell lines (> 15% increase in cytochrome c loss). By measuring ROS levels, we found increased ROS production (> 2-fold increase in median fluorescent intensity) in response to JNJ-64619178 before the onset of apoptosis, suggesting a possible mechanism by which JNJ-64619178 increases apoptotic priming. Conclusions: The combination of a PRMT5 inhibitor with BH3 mimetics, especially venetoclax, is worthy of further exploration as a potential therapeutic strategy for DLBCL and MCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IRF4 Shapes the Tumor Microenvironment of Follicular Lymphoma

Follicular lymphoma (FL) is the most common indolent B cell lymphoma with a typically favorable outcome. However, ~20% of these patients have an early relapse with a poor prognosis. Thus far, efforts to identify factors that predict survival have been unsuccessful. However, we and others have shown the prognostic relevance of the tumor microenvironment (TME) and provided initial evidence for the role of specific genetic alterations in shaping different environments with highly dissimilar clinical courses. We have also identified three distinct molecular clusters, one of which was characterized by an IRF4 signature, an unfavorable TME and a poor outcome. The master regulator IRF4 plays a critical role in terminal B cell differentiation. To investigate whether IRF4 modulates immune signatures in the TME, we integrated RNA-seq, mass cytometry (CyTOF) and whole exome sequencing (WES) of 88 newly diagnosed FL grade 1-3 patients. Using an established gene set that identifies the IRF4 signature(Shaffer et al, Nature 2008), we first performed singscore gene set scoring to discriminate IRF4high and IRF4low categories. IHC analysis of IRF4 expression in 20 samples with IRF4 high and low transcriptional signature validated this classification, with excellent concordance. Using CIBERSORT, we found that FLs with IRF4high expression had a strikingly different TME compared to IRF4low tumors. Specifically, there was a significant enrichment for CD4+ T cells (p<0.001), Treg cells (p<0.001), as well as M1 (p=0.027) and M2 macrophages (p<0.001, A). In line with the transcriptomic data, CyTOF analysis performed in 85 out of 88 FL cases showed that CD4+ (p=0.01), Treg (p=0.01) and TFH (p=0.03) cells differed significantly in IRF4high vs IRF4low FLs. Moreover, a CITRUS cluster analysis identified three significantly different clusters, including TFH exhausted and Treg cells. RNA-seq of these 88 FL tumors revealed distinct transcriptomic profiles (FDR<0.05) according to IRF4 status with upregulation of 3,448 genes and downregulation of 2,907 genes. Pathway analysis using the Molecular Signature Database (MSigDB) revealed striking enrichment for (i) direct IRF4 targets (FOXP3, FOXA1); (ii) NF-kB targets (TP53, TLR signaling); and (iii) signatures associated with immune signaling including co-stimulatory (CD40 signaling) and inhibitory signaling (PD1 signaling), antigen presentation (MHC class I and II), upregulated Treg and downregulated TFH signatures (B). To investigate the somatic mutations and copy number alterations associated with differential IRF4 expression, we performed WES of purified B cells from the same 88 FLs and further expanded this cohort for a total 126 FLs. We found enrichment for mutations in B2M (which is a component of MHC class I) in IRF4high vs IRF4low FLs (p=0.024). The same cases showed a negative correlation for CREBBP loss-of-function mutations (which modulates MHC class II; p=0.008). Silencing of IRF4 in TMD8 and SUDHL2 cells (ABC lymphoma cells with high IRF4 expression) by small interference RNA (siIRF4) increased CD40 and MHC class II expression while downregulating PDL1 at the transcriptional and protein levels (C). Conversely, ectopic expression of IRF4 in OCI-LY19 cells (GCB lymphoma cells with low IRF4 expression) generated the opposite effect. Using a luciferase-based promoter assay, we ruled out NF-kB-mediated control of IRF4 effects, as selective inhibition of IRF4 did not alter NF-kB luciferase activity. To investigate whether IRF4 silencing could trigger anti-lymphoma immunity, we co-cultured CD4+ T cells with TMD8 and SUDHL2 cells transfected with siIRF4 or siCtr for 5 days. As expected, IRF4 knockdown in these cells resulted in decreased cell viability, however this cytotoxic effect was significantly increased in the presence of CD4+ T cells, consistent with T cell-direct tumor killing (D). Notably, silencing of IRF4 in lymphoma cells doubled the number of TFH cells while halving the Treg cells compared to siCtr (E). In support of the T cell activation, ELISpot for IFNγ showed 8-10-fold increase when B cells were transfected with siIRF4. Collectively, we found that FLs with IRF4high expression are associated with a suppressive TME and selective IRF4 silencing restores anti-lymphoma T cell immunity. Further investigation is warranted to identify the mechanisms by which IRF4 controls tumor immunity to develop precision therapies for this unfavorable population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting Sumoylation with Subasumstat (TAK-981), a Selective Inhibitor of SUMO-Activating Enzyme (SAE), Induces Mitochondrial Dysfunction in Preclinical Models of Non-Hodgkin Lymphoma (NHL)

SUMOylation is a post-translational modification which leads to covalent attachment of small ubiquitin-like modifiers (SUMO) proteins to lysine residues of a target protein. SUMOylation is essential for the regulation of genomic integrity, gene expression and intracellular signaling. Many tumors exhibit deregulation of SUMOylation pathway. Subasumstat, (TAK-981), a novel selective inhibitor of SUMO-activating enzyme (SAEi), has entered early phase II clinical trials. Here we studied SAEi in pre-clinical models of B-cell non-Hodgkin's lymphoma (NHL). TAK-981 was provided by Takeda Development Center Americas, Inc. (Lexington, MA). Diffuse large B-cell lymphoma (DLBCL) cell lines (OCI-LY3, VAL, U-2932, SU-DHL10), and primary mantle cell lymphoma (MCL) cells were assayed for survival, mitochondrial function, and metabolic phenotype. In vivo experiments were performed using DLBCL cell line xenografts and MCL PDX models. Treatment with TAK-981 restricted growth of DLBCL cell lines with an IC50~10 nM. SAEi led to rapid (within 2 h) protein desumoylation in a dose dependent manner, prominently in the ~90 kDa region. TAK-981 induced pronounced double strand DNA breaks in DLBCL cells (comet assay) and cell cycle arrest (propidium iodine). RNA-Seq and ATAC-Seq analysis of TAK-981-treated DLBCL cells revealed upregulation of the DNA damage, NF-κB, and OXPHOS signaling pathways. We noted that in addition to cytoplasmic proteins, mitochondrial proteins were heavily de-SUMOylated upon treatment of NHL cells with TAK-981. In vitro exposure to TAK-981 resulted in mitochondrial membrane depolarization, loss of mitochondrial integrity (by electron microscopy) and a change of mitochondrial dynamics as assessed by expression of mitochondrial proteins (eg, Drp1). Seahorse respirometry analysis revealed dramatic downmodulation of OXPHOS following SAEi. Furthermore, metabolomic profiling of DLBCL cells treated with TAK-981 showed downmodulation of TCA substrates, while glycolysis substrates remained unchanged. The mitochondrial disruption observed following TAK-981 treatment was accompanied by rapid accumulation of reactive oxygen species, a mechanism that has been previously shown to lead to DNA damage in cancer cells. Consistent with cell line data, exposure of primary MCL cells to TAK-981 in stromal conditions (CD40L- or BAFF expressing stroma) induced apoptosis and reduced OXPHOS. Next, we conducted genome-wide CRISPR-Cas9 loss-of-function library screens and identified that loss of genes in the NFκB, TP53, DNA damage and centromere/telomere gene pathways contributed to resistance to SAEi in DLBCL cells. By means of genetic knockout we demonstrated that TP53 and its transcriptional target BAX contributed to TAK-981-induced G2 arrest and apoptosis, further implicating mitochondrial dysfunction in sensitivity to SAEi. For in vivo experiments, we used DLBCL xenografts. OCI-LY3 and U-2932 cells were inoculated subcutaneously in NSG mice. Once tumors reached 100 mm3, mice received 10 twice-weekly IV doses of 7.5 mg/kg TAK-981 (over 5 weeks), leading to resolution of flank tumors and improved survival compared with control. Finally, we used an MCL PDX model where MCL cells were inoculated intravenously. Once cells became detectable in the blood, mice were treated as above. Treatment with TAK-981 resulted in delayed tumor expansion in blood and spleen and extended animal survival. TAK-981-exposed splenocytes exhibited a reduction of maximal respiration by Seahorse analysis. Conclusions. Pharmacologic SAEi with TAK-981 demonstrated pre-clinical activity in NHL models in vitro and in vivo, accompanied by DNA damage, mitochondrial dysfunction, and metabolic reprogramming.

ULK1 Blockade Preferentially Targets Germinal Centre B-Cell - Diffuse Large B-Cell Lymphoma Subtype By Attenuating Autophagy, c-MYC and Inflammation

Diffuse Large B-cell Lymphoma (DLBCL) is a heterogeneous lymphoid malignancy. Gene Expression profiling (GEP) has characterized two distinct subsets: Germinal Centre B-cell (GCB) and Activated B-cell (ABC) DLBCL. Oncogenic PI3K-AKT signaling has been identified in primary DLBCL subtypes[1]. PI3K-AKT is activated in response to the loss of PTEN in GCB-DLBCL. In ABC-DLBCL the activation is associated with CD79B mutations and concomitant MYD88 alterations. The Bruton Tyrosine Kinase (BTK) functions as an intermediary kinase. Ibrutinib (BTK inhibitor) has demonstrated promising capability in ABC-DLBCL unlike GCB tumors, warranting further investigations[2]. PI3K-AKT-mTOR signaling is also a known regulator of macro-autophagy (herein referred to as autophagy). ULK1 complex is critical for the initiation of autophagy. Aberrant signaling in this pathway promotes nutritional adaptation to malignant cells and contributes to drug resistance. We hypothesize autophagy facilitates cytoprotection and contributes to drug resistance including R-CHOP and Ibrutinib in GCB-DLBCL. Dual targeting of the BTK with Ibrutinib and autophagy initiating complex(es) would be a strategic method to impair these oncogenic functions. To determine the presence of increased autophagy related genes (ATGs) and its association to R-CHOP treatment outcome, we analyzed the GEP of patients from the REMODL-B trial (R-CHOP: GCB n=240 and ABC n=121)[3]. Gene set enrichment analysis (GSEA) showed no difference between the DLBCL patients indicating mutual autophagy dependency. Genes associated to worst overall survival (OS) in autophagy initiating complex I/ ULK1 complex (ULK1, RB1CC1/FIP200 and ATG13) and complex II (AMBRA1, PIK3C3/VPS34, PIK3R4/VPS15 and USO1/p115) were identified using Kaplan-Meier curves and overlapped according to their respective complexes. Statistical analysis confirmed ULK1 complex was significantly dominate in response to R-CHOP treatment leading to poor OS (p=0.0403). These findings provide a rational to inhibit autophagy via the ULK1 complex in GCB patients. Alike, multiple GCB (n=7) and ABC (n=5) cell lines showed substantial autophagy dependency using LC3B autophagy marker expression. Ibrutinib treatment enhanced autophagic flux in GCB cell lines. Addition of ULK1 complex inhibitor (MRT68921) augmented the anti-tumor activity of Ibrutinib in HT, Oci-Ly1, Oci-Ly18 and SUDHL-6 GCB cell lines and activated caspase dependent apoptosis. Interestingly, ULK1 complex inhibitors (MRT68921/ ULK1-101) induced acute NF-ҡB activation in multiple GCB and ABC lymphoma lines irrespective of their NF-ҡB mutational status. RNA sequencing of GCB and ABC cell lines treated with MRT68921, or vehicle reported diverse ULK1 inhibitory signaling pathways. Oci-Ly1 (GCB, c-MYC amplification) treated cells significantly downregulated genes associated to PI3K-mTOR activity/ signaling, autophagy, metabolism, and IL-6. GSEA reported a substantial decrease in c-MYC transcriptional activation/ pathway and TNF signaling. Immunoblotting data confirmed ULK1 inhibition dephosphorylated c-myc(ser62) in a time dependent manner in Oci-Ly1 and Oci-Ly18. In contrast, Oci-Ly3 (ABC, c-MYC amplification) treated cells upregulated genes involved in JAK-STAT pathway, IL-6, and apoptosis. C-MYC remained unaffected. In summary, GCB-DLBCL patients treated with R-CHOP harbor increased autophagy signatures that potentially dictate treatment outcome. Our in vitro data demonstrates ULK1 inhibition sensitizes GCB cell lines to Ibrutinib due to diverse role in regulating NF-ҡB, c-MYC and inflammation. Unlike, ABC, where ULK1 blockade promotes immunogenic-dependent cell death.

Antigen-Independent, Autonomous B-Cell Receptor Signaling in Diffuse Large B-Cell Lymphoma

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) defines the Cell-of-origin (CoO) classification with two major subtypes, germinal center B-cell (GCB) and the more aggressive activated B-cell (ABC) type. Chronic activation of B-cell receptor (BCR) signaling with constitutive activation of the NF-κB pathway is an essential oncogenic mechanism in ABC DLBCL. BCR pathway activation with increased NF-κB activation can be caused by gain-of-function mutations of the BCR signaling cascade. We investigated whether antigen-independent, i.e. autonomous BCR signaling, akin to its essential pathogenetic role in chronic lymphocytic leukemia, acts as a functional driver mechanism in DLBCL without such gain-of-function mutations. DNA and RNA were extracted from ABC DLBCL cell lines TMD8 and OCI-Ly3 and from fresh-frozen biopsies of 18 histologically confirmed DLBCL cases. Functional BCR transcripts were identified by ARTISAN PCR and transduced into murine triple knock out (TKO) cells. TKO cells lack the recombination activation gene 2 (Rag2) and lambda 5 gene (λ5) and are therefore incapable of expressing an endogenous BCR. In addition, the BCR signal transduction molecule SLP65 is replaced with a tamoxifen-inducible estrogen receptor (ERT2) - SLP65 fusion gene. Upon transduction of both heavy and light BCR chains and staining with Indo-1, background calcium flux can be measured as Indo-1 signal intensity ratio (SIR) by flow cytometry. After restoring functionality of the BCR signaling cascade by tamoxifen, antigen-independent BCR signaling is measured as SIR, followed by maximum BCR stimulation with an isotype-specific crosslinking antibody. Autonomous BCR signaling strength (SSBCR) is quantified by an arithmetic product based on the relative Indo-1 SIR and the relative number of cells with SIR above background in the presence of tamoxifen and calibrated on crosslinking-induced maximum SIR. According to the primary hypothesis, the BCR of CARD11 wild-type, IgM-expressing TMD8 cells demonstrated strong autonomous BCR signaling (SSBCR=0.243), whereas the BCR of CARD11 mutated, IgG-expressing OCI-Ly3 cells completely lacked autonomous signaling (SSBCR=0.022). BCR from 13 primary DLBCL signaled autonomously (SSBCR=0.125-0.619), where 5 samples were negative (SSBCR=0.004-0.023) Autonomous BCR signaling was absolutely dependent on IgM isotype (Fisher's exact test: p=0.0016). CoO classification by digital gene expression (Nanostring) analyses or immunohistochemistry demonstrated autonomous BCR signaling in 8/9 ABC (non-GCB) DLBCL, 2/6 GCB DLBCL, and 2 unclassifiable DLBCL, and a transformed follicular lymphoma DLBCL. Independent of CoO, DLBCL can be assigned to 6-7 clusters based on their genetic aberrations (Chapuy 2018, Wright 2020). Fifteen DLBCL were suited for WES and targeted panel sequencing. Four cases were assigned by a probabilistic calculation to Chapuy cluster C5, 2 cases to C2, and one case to C1, while 8 cases remained unclassifiable. Clustering by the LymphGen tool assigned 3 cases to cluster ST2, one case each into MCD, EZB, and N1, 4 cases into various clusters, and 5 cases unclassified. DLBCL with autonomous BCR signaling fell into clusters C1/unclassified, C2/unclassified, C5/unclassified (n=3), unclassified/EZB, unclassified/ST2, unclassified/N1 and unclassified/unclassified (n=2). Cases lacking autonomous BCR signaling were assigned to unclassified/ST2 (n=2), C5/MCD, and unclassified/unclassified. Eleven of 13 cases with an autonomous signaling BCR lacked activating CARD11 mutations. In conclusion, autonomous BCR signaling is a potential immunological oncogenic driver in DLBCL that cannot be detected by sequencing studies, is independent of external antigen stimulation, and originates from a self-organizing conformation of the surface BCR (Minici 2017). Autonomous BCR signaling is present predominantly in ABC DLBCL and dependent on IgM isotype. Autonomous BCR signaling offers a convincing explanation for oncogenic NF-κB activation in DLBCL. Limited data suggest that the majority of DLBCL with autonomous BCR signaling fall into the ABC-dominated genetic clusters C1/C5, and MCD/N1, respectively. In summary, genetic aberrations, BCR function, and gene expression profile form 3 independent layers of DLBCL pathophysiology that need to be analyzed separately and offer independent targets for therapy.

Efficacy Analysis of Bruton's Tyrosine Kinase Inhibitor Combined Regimen in the Treatment of Elderly Patients with Relapsed/Refractory Primary Central Nervous System Lymphoma

Background Primary central nervous system lymphoma (PCNSL) is a kind of aggressive non-Hodgkin lymphoma that originates in the brain, spinal cord, eyes or pia mater without evidence of systemic involvement, which accounts for about 3% of central nervous system tumors and has a poor prognosis. Most patients will eventually relapse and the 5-year survival rate is 29.6%. Zanubrutinib is a kind of new generation BTK inhibitor, which can penetrate the blood-brain barrier and be used in the treatment of PCNSL. The study aims to investigate the efficacy and safety of a zanubrutinib-based combination regimen in the treatment of elderly relapsed/refractory(R/R) PCNSL. Methods We retrospectively analyzed elderly patients with R/R PCNSL who received zanubrutinib-based combination regimen at the Affiliated Hospital of Nantong University over the past few years. We collected the information of patients diagnosed with PCNSL in the SEER project of the National Cancer Institute (NCI), and analyze the survival status. We used CCK8 and EdU to detect the effect of zanubrutinib on the viability and proliferation of activated B-cell subtype (ABC) diffuse large B-cell lymphoma (DLBCL) cell lines with genotypes similar to PCNSL. Results The 7 elderly R/R PCNSL patients treated with zanubrutinib-based regimen (zanubrutinib, rituximab and temozolomide) showed an overall response rate (ORR) of 85.7% (6/7), a complete response (CR) of 57.1% (4/7) and a partial response (PR) of 28.6% (2/7), and 14.3% (1/7) of patients experienced disease progression (PD). Patients' gender, KPS score, ECOG score, surgical treatment or not, positive cerebrospinal fluid protein (all P>0.05) were not significantly associated with the treatment effect. Pathological type and IESLG score were related to treatment effect (P<0.05). The median progression-free survival (PFS) and median overall survival (OS) of patients in this study were not reached. Through the analysis of clinical data of PCNSL patients in SEER database from 1975 to 2016, it is concluded that elderly patients (age>60 years old) and male PCNSL patients have a worse prognosis, the primary site of PCNSL, treatment methods and the choice of the disease have impact on the prognosis, and there is no significant difference between the disease and other causes in terms of the cause of death. In the survival analysis of elderly patients, gender, surgery and radiotherapy sequence had no significant effect on prognosis, and patients who received chemotherapy had better OS than those who received radiotherapy. The cell viability of ABC subtype DLBCL cell line OCI-Ly10 decreased in a concentration- and time-dependent manner with zanubrutinib and temozolomide (P<0.05). The inhibitory effect of zanubrutinib and temozolomide on the proliferation of OCI-Ly10 cell line was stronger than that of single drug. Conclusion Zanubrutinib-based combination therapy has good efficacy and controllable safety in elderly R/R PCNSL patients. The prognosis of elderly patients is poor, and the OS of chemotherapy patients is better than that of radiotherapy among the treatment options in the pre-BTKi era. Zanubrutinib inhibits cell viability and cell proliferation in ABC subtype DLBCL cell line OCI-Ly10.

Antitumor Effect of Dihydroartemisinin on Diffuse Large B-Cell Lymphoma

To investigate the potential antitumor effect and its mechanism of dihydroartemisinin (DHA) on diffuse large B-cell lymphoma (DLBCL). OCI-Ly7 cells were respectively treated with different concentrations of DHA (0, 12.5, 25, 50 and 100 μmol/L) , CCK-8 was used to detect the cells viability. Subsequently, OCI-Ly7 cells were divided into 5 groups : DHA 0,25,50,100 μmol / L and DHA (100 μmol / L) + Colivelin (STAT3 activator). Aldehyde dehydrogenase (ALDH) positive cells were sorted by flow cytometry, the sphere-forming ability of stem cells was detected. Transwell assay and scratch test were used to analyze the invasion and migration of cells. Western blot was used to detect the expression of migration and invasion-related proteins, as well as the phosphorylation levels of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3(STAT3). DHA induced obvious cytotoxicity to OCI-Ly7 cells. Compared with the control group, the stem cell-like properties, invasion and migration of OCI-Ly7 were significantly inhibited in DHA 50 μmol/L group and 100 μmol/L group, while the phosphorylation levels of JAK2 and STAT3 were significantly reduced. There was no significant difference in DHA 25 μmol/L group compared with the control group. Treated with Colivelin, the inhibition of DHA on OCI-Ly7 stem cell-like properties, invasion and migration was significantly reversed, and the expression of p-STAT3 was significantly up-regulated. DHA has antitumor effect on DLBCL, and its mechanism may be through inhibiting the activation of JAK2/STAT3 pathway to inhibit the stem cell-like properties, invasion and migration of DLBCL cells.

Abstract A02: Probing the roles of SUMOylation in B-cell lymphoma cells by using a selective SUMO inhibitor subasumstat (TAK-981)

Abstract SUMOylation is a post-translational modification that leads to covalent attachment of small ubiquitin-like modifiers (SUMO) proteins to a target protein. SUMOylation is essential for the regulation of genomic integrity, gene expression and intracellular signaling. Many tumors exhibit deregulation of SUMOylation pathway. TAK-981, a novel selective inhibitor of SUMO-activating enzyme (SAEi) has entered early phase II clinical trials. Here we studied SAEi in pre-clinical models of non-Hodgkin lymphoma (NHL). TAK-981 was provided by Takeda (Lexington, MA). Diffuse large B-cell lymphoma (DLBCL) cell lines and primary mantle cell lymphoma (MCL) cells were assayed for survival, mitochondrial function and metabolic phenotype. In vivo experiments were performed using DLBCL cell line xenografts and MCL PDX models. Treatment with TAK-981 restricted growth of DLBCL lines and led to rapid protein de-SUMOylation in a dose-dependent manner. TAK-981 induced pronounced DNA break in DLBCL cells (comet assay) and cell cycle arrest (propidium iodide). RNA-Seq and ATAC-Seq analysis of TAK-981-treated OCI-LY3 and U-2932 cells revealed upregulation of the NFκB, and OXPHOS signaling pathways. We noted that mitochondrial proteins were heavily de-SUMOylated upon treatment of NHL cells with TAK-981. In vitro exposure to TAK-981 resulted in mitochondrial membrane depolarization, loss of mitochondrial integrity (EM) and a change of mitochondrial dynamics (ICC for p-Drp1), accompanied by rapid accumulation of reactive oxygen species. Seahorse respirometry revealed dramatic downmodulation of OXPHOS. Metabolomic profiling of DLBCL cells treated with TAK981 exhibited downmodulation of TCA substrates. Exposure of primary MCL cells to TAK-981 in stromal conditions (CD40L- or BAFF expressing stroma) induced apoptosis and reduced OXPHOS. Next, we conducted genome-wide CRISPR-Cas9 loss-of-function library screens and identified that loss of genes in the NFκB, TP53 and DNA damage pathways contributed to resistance to SAEi in DLBCL cells. By means of genetic knockout we demonstrated that TP53 and its transcriptional target BAX contributed to TAK-981-induced G2 arrest and apoptosis, further implicating mitochondrial dysfunction in sensitivity to SAEi. For in vivo experiments, OCI-LY3 and U-2932 cells were inoculated subcutaneously in NSG mice. Once tumors reached 100 mm3, mice received twice-weekly IV doses of 7.5 mg/kg TAK-981 (over 5 weeks), leading to resolution of flank tumors and improved survival compared with control. Finally, we used an MCL PDX model where MCL cells were inoculated intravenously. Once cells became detectable in the blood, mice were treated as above. Treatment with TAK-981 resulted in delayed tumor expansion in blood and spleen and extended animal survival. TAK-981 exposed splenocytes exhibited a reduction of maximal respiration by Seahorse. In summary, pharmacologic SAEi with TAK-981 demonstrated pre-clinical activity in NHL models in vitro and in vivo, accompanied by DNA damage, mitochondrial dysfunction and metabolic reprogramming. Citation Format: Tingting Liu, Vi Lam, Duanchen Sun, Elana Thieme, Tamilla Nechiporuk, Daniel Bottomly, Olga V Danilova, Nur Bruss, Martina Cusan, Lili Wang, Shannon K McWeeney, Jeffrey W Tyner, Steve Kurtz, Zheng Xia, Alexey Danilov. Probing the roles of SUMOylation in B-cell lymphoma cells by using a selective SUMO inhibitor subasumstat (TAK-981) [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A02.

Abstract A29: A selective HDAC3 inhibitor synergizes with 5-azacytidine in diffuse large B-cell lymphoma

Abstract Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma, with 40% of patients relapsing or refractory to the conventional chemotherapy treatment, usually with fatal consequences. Therefore, more targeted therapies are needed. DLBCL are characterized by profound alterations in the epigenome (histone modifications and DNA methylation) that are correlated with poor survival. While epigenetic drugs are used as anti-cancer treatments, their full potential has not been achieved. One limitation for the implementation of epigenetic-based therapies in the clinic has been its use as single agents; therefore, we explored new combinatorial therapies with the goal to increase efficacy. Our previous studies showed that lymphoma epigenetic programming involves repression of BCL6 target genes. This BCL6-mediated gene silencing in DLBCL is driven by 1) reduced H3K27 acetylation through recruitment of histone deacetylase 3 (HDAC3) and 2) reduced 5-hydroxymethylcytosine (5hmC). We thus tested a therapy consisting of the hypomethylating agent 5-azacytidine (5-Aza) and a specific HDAC3 inhibitor (HDAC3i). DLBCL cell line MD901 was treated with 200nM 5-Aza or vehicle daily for 5 days, with 10uM HDAC3i or vehicle added at days 3 and 5. Analysis at day 8 showed that whereas single agent did not induce cell death based on propidium iodide (PI) staining (vehicle=13.5%, 5-Aza=18%, HDAC3i=14.8% PI+ cells), the combination induced significantly higher cell death (5-Aza+HDAC3i=43.6% PI+ cells). We observed similar increased efficacy of 5-Aza+HDAC3i in OCI-Ly7 and SUDHL-4 cells. We also demonstrated that 5-Aza+HDAC3i acted synergistically in MD901, OCI-Ly7 and SUDHL-4 (combination index (CI)&amp;lt;1), with a dose-reduction index (DRI)=3.1 for 5-Aza and DRI=2.6 for HDAC3i to achieve 50% cell death in MD901; similar results were obtained in the other cell lines tested. We established xenografts -transplanting lymphoma cells in NSG mice through intraperitoneal injection- to test the efficacy of the combinatorial therapy in vivo. Mice engrafted with OCI-Ly7 cells received daily treatment with 1mg/kg 5-Aza or vehicle for 1 week, and were also treated with 25mg/kg HDAC3i or vehicle for other 3 weeks. 80% mice treated with 5-Aza+HDAC3i survived after 150 days, compared to 50% mice treated with HDAC3i alone; 5-Aza did not induce survival advantage compared to vehicle-treated mice. At the molecular level, we found that 5-Aza as single agent reduced global 5hmC levels compared to vehicle-treated cells; interestingly, this was also observed when cells were treated with HDAC3i alone. Intriguingly, the level of 5hmC when combining 5-Aza+HDAC3i was comparable to vehicle-treated cells. In summary, we demonstrate superior efficacy of 5-Aza in combination with HDAC3i in DLBCL. We are currently performing genome-wide assays to dissect the molecular mechanisms underpinning this synergistic anti-tumor activity. At the same time, we are generating xenograft models with primary DLBCL samples to confirm the potential of this new therapeutic approach to improve the outcomes of DLBCL patients. Citation Format: Pilar M Dominguez, Leonie A Cluse, Noura Tawfic, Peter J Fraser, Sophia Rutaquio, Rachel Lim, Giorgio Inghirami, Gareth Gregory, Michael Dickinson, Ari M Melnick, Ricky W Johnstone. A selective HDAC3 inhibitor synergizes with 5-azacytidine in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A29.

Knockdown of the long non-coding RNA CACNA1G-AS1 enhances cytotoxicity and apoptosis of human diffuse large B cell lymphoma by regulating miR-3160-5p.

: Long non-coding RNAs (lncRNAs) have been confirmed to be connected with tumor proliferation, apoptosis, metastasis and recurrence. Previous studies have indicated that lncRNA calcium voltage-gated channel subunit α1 G (CACNA1G)-antisense 1 (AS1) can function as a pro-oncogene in several types of cancer. However, the specific role and mechanism of CACNA1G-AS1 have not been fully elucidated in human diffuse large B cell lymphoma (DLBCL). In the present study, CACNA1G-AS1 expression was verified in DLBCL tissues and cells by reverse transcription-quantitative PCR, and the relationship between CACNA1G-AS1 and microRNA (miR)-3160-5p was confirmed using luciferase reporter assays. After CACNA1G-AS1-knockdown and miR-3160-5p-overexpression, MTT, colony formation and flow cytometry assays were conducted to assess the changes in the cytotoxicity and apoptosis of OCI-Ly10 and SUDHL-4 cells. In addition, in vivo experiments were performed to determine the impact of CACNA1G-AS1-knockdown on tumor growth and apoptosis. It was revealed that CACNA1G-AS1 was highly expressed in DLBCL tissues and cells and that expression of CACNA1G-AS1 was associated with the clinical stage of DLBCL. Functionally, CACNA1G-AS1-knockdown was demonstrated to increase cytotoxicity and expedite apoptosis in DLBCL cells in vitro and in vivo. In addition, CACNA1G-AS1 could downregulate miR-3160-5p by targeting binding in DLBCL cells. Overexpression of miR-3160-5p had the same effects on the cytotoxicity and apoptosis of DLBCL cells as CACNA1G-AS1-knockdown. Overall, the present study revealed that CACNA1G-AS1-knockdown and miR-3160-5p-overexpression could prevent DLBCL carcinogenesis, which might provide novel therapeutic targets for DLBCL.

Effects of BET Bromodomain Inhibitor JQ1 on Double-Expressor Lymphoma Cell Lines and Its Mechanism

To investigate the effects and mechanism of bromodomain and extra-terminal (BET) inhibitor JQ1 on the double-expressor lymphoma (DEL) cell lines. Protein expressions of cMyc and BCL-2 in 3 lymphoma cell lines were checked by Western blot so as to identify DEL cell lines. CCK-8 assay was used to detect the effects of JQ1 on anti-proliferation in the DEL cell lines. Western blot and RT-PCR were used to measure the protein and mRNA expressions of cMyc, BCL-2 and BCL-6 in DEL cell lines which treated by JQ1. Flow cytometry was used to detect the effect of JQ1 on cell apoptosis. Based on the expressions of cMyc and BCL-2, the SU-DHL6 and OCILY3 cell lines were confirmed as DEL cell lines. CCK-8 assay showed that the proliferation of DEL cell lines was inhibited by JQ1, which was similar to non-DEL cell lines and mainly regulated the expression of cMyc and BCL-6 but not BCL-2. JQ1 had no effects on apoptosis in the DEL cell lines. BET inhibitor JQ1 has anti-tumor effect in the DEL cell lines, thus providing evidence for the therapeutic potential of BET inhibitor JQ1.

Abstract 1400: AS-1763: a highly potent, noncovalent and next generation BTK inhibitor for the treatment of patients with B-cell malignancies having C481S mutation in BTK

Abstract Introduction: Bruton's tyrosine kinase (BTK) has been recently recognized as a validated drug target for the treatment of B-cell malignances. The emergence of clinical resistance to the first-generation covalent BTK inhibitors is, however, becoming a serious concern. Patients are reported to develop resistance during the treatment due to substitution of cysteine residue at position 481 with serine (C481S mutation) in BTK, which prevents the covalent binding of the first-generation irreversible BTK inhibitors, resulting in the loss of inhibitory activities. Therefore, there is a high unmet medical need for new therapeutic approaches to overcome the BTK C481S-mediated resistance. Material and methods: Compounds were tested in biochemical assays using recombinant human wildtype (WT) and C481S mutant BTKs. Cellular activity of AS-1763 was evaluated in human diffuse large B cell lymphoma (DLBCL) cell lines, OCI-Ly10 and its BTK[C481S] knock-in cells. The in vivo antitumor activity of AS-1763 was assessed in two tumor xenograft mouse models bearing WT or BTK [C481S] knock-in OCI-Ly10 cells. Results: We have discovered AS-1763 as an orally available, highly potent, selective and reversible BTK inhibitor. AS-1763 showed potent inhibitory activities for WT BTK (an activated form, BTK[A]) and the C481S mutant with sub-nanomolar IC50s (IC50 = 0.85 and 0.99 nM for BTK[A] and BTK[C481S], respectively). In cellular assays, AS-1763 exhibited strong antiproliferative activities for WT and BTK [C481S] knock-in OCI-Ly10 cells. AS-1763 demonstrated significant tumor growth inhibition and tumor regression in WT as well as BTK[C481S] knock-in OCI-Ly10 xenograft model. Conclusions: The preclinical data support the clinical development of AS-1763 in patients with B-cell malignancies both having WT and C481S mutation in BTK. The first-in-human Phase 1 clinical trial of AS-1763 in healthy volunteers (EudraCT 2020-005599-37) is currently ongoing, and the Phase 1b study in patients is expected to begin in 2022. Citation Format: Wataru Kawahata, Tokiko Asami, Takao Kiyoi, Takayuki Irie, Shigeki Kashimoto, Hatsuo Furuichi, Masaaki Sawa. AS-1763: a highly potent, noncovalent and next generation BTK inhibitor for the treatment of patients with B-cell malignancies having C481S mutation in BTK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1400.

Abstract 1879: A novel implantable device to in vivo assess anti-cancer agents

Abstract Background: The high variability in clinical responses observed in cancer patients highlights the need of a tailored therapeutic approach. A possible modality is to assess drugs sensitivity directly in the patients, by introducing small drug delivering devices in tumor sites for a very short period and then looking at the local anti-tumor effect. Here, we present the design of an innovative drug eluting device and its test with the BTK inhibitor ibrutinib as an example of small molecules. Methods: Mathematical models considered factors related to drug (MW), physical properties, desired concentrations in surrounding tissue, polymers and tissue physical features to identify the optimal polymers and the drug loading for the desired release profile over 24h. In vitro proliferation was measured with the MTT assay, in vivo experiments done in NOD-SCID mice (license TI05/2021), and immunohistochemistry on FFPE xenograft sections stained for Ki67 and cleaved caspase 3 (CASP-3). Results: Device was designed as an arrow-shaped cylinder, with flat end and flatter sections to be filled with the drug-eluting polymers. Prototypes were built in nylon6,6, a biocompatible but stable polymer. Ibrutinib was incorporated in low MW polyester poly-ε-caprolactone (PCL) as biopolymer by solvent casting. Polymeric coating onto devices was done with a dedicated automatic device.Devices loaded with biopolymer and different concentrations of ibrutinib or “empty” biopolymers were first in vitro tested using diffuse large B cell lymphoma (DLBCL) cell lines. Over 72h, devices with drug inhibited proliferation of the ibrutinib-sensitive TMD8 and OCI-Ly10 cell lines, but not of the ibrutinib-resistant SU-DHL-2 and U2932. No effect was seen with devices with ibrutinib-free biopolymers. Devices, empty or loaded with ibrutinib (5μg), were then inserted in xenografts of ibrutinib-sensitive cell line OCI-Ly10 and ibrutinib resistant U2932. After 24h, mice were sacrificed and xenografts analyzed. By Ki67 and CASP-3 a reduced cell proliferation and an increased apoptosis in the region surrounding the device was observed in the ibrutinib-sensitive xenografts, conversely nor reduced cell proliferation nor apoptosis induction were identified in the ibrutinib-resistant xenografts. Conclusions: We have created a prototype of a device that can locally release drugs allowing the evaluation of anti-tumor activity, optimizing cures tailored to single patient. The system can be further developed to include multiple drugs, including e.g. antibodies. Citation Format: Giuseppe Perale, Eugenio Gaudio, Tommaso Casalini, Luca Aresu, Anna Rita De Corso, Filippo Spriano, Chiara Tarantelli, Anastasios Stathis, Andrea Castrovinci, Francesco Bertoni. A novel implantable device to in vivo assess anti-cancer agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1879.

IDO1 plays a tumor-promoting role via MDM2-mediated suppression of the p53 pathway in diffuse large B-cell lymphoma

With the intensive therapeutic strategies, diffuse large B-cell lymphoma (DLBCL) is still a fatal disease due to its progressive characteristics. Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulator that catalyzes the commitment step of the kynurenine pathway in the immune system, its aberrant activation may contribute to malignant cell escape eradication. However, the role of IDO1 in DLBCL progression remains elusive. Our study showed IDO1 expression was upregulated in DLBCL and was associated with a poor prognosis and low overall survival. Inhibition of IDO1 suppressed DLBCL cell proliferation in vitro and impeded xenograft tumorigenesis in vivo. RNA-seq analyses revealed MDM2 was downregulated while TP53 was upregulated in IDO1 inhibition OCI-Ly10 cells. Mechanistically, IDO1 inhibition decreased the expression of MDM2, a major negative regulator of p53, and restored p53 expression in OCI-Ly3 and OCI-Ly10 cells, resulting in cell cycle arrest and apoptosis. IDO1 inhibition induced cell apoptosis coupled with PUMA and BAX upregulation, as well as BCL2 and BCL-XL downregulation. In addition, p21, a p53 transcriptional target, was upregulated in cell cycle arrest. Taken together, this study revealed IDO1 is essential for the proliferation of DLBCL cells and may be a potential therapeutic target for the treatment of DLBCL.

Identification of LINC00654-NINL Regulatory Axis in Diffuse Large B-Cell Lymphoma In Silico Analysis.

BackgroundThe long non-coding RNA (lncRNA)-mRNA regulation network plays an important role in the development of diffuse large B-cell lymphoma (DLBCL). This study uses bioinformatics to find an innovative regulation axis in DLBCL that will provide a positive reference for defining the mechanism of disease progression.MethodsBatch Cox regression was used to screen prognosis-related lncRNAs, and a random forest model was used to identify hub lncRNA. The clinical value of the lncRNA was evaluated and Spearman correlation analysis was used to predict the candidate target genes. Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were used to define the biological function of the lncRNA. A batch Cox regression model, expression validation, and Spearman correlation analysis were used to select the best downstream target genes. The expression and prognostic value validation of this gene was conducted using public data. Gene Set Enrichment Analysis (GSEA) was performed to explore potential mechanisms for this gene in DLBCL.ResultsLINC00654 was identified as the hub lncRNA and 1443 mRNAs were selected as downstream target genes of the lncRNA. The target genes were enriched in the regulation of GTPase and Notch signaling pathways. After validation, the ninein-like (NINL) gene was selected as the potential target of LINC00654 and the LINC00654-NINL axis was constructed. Patients with better responses to therapy were shown to have high NINL gene expression (p-value = 0.036). NINL also had high expression in the DB cell line and low expression in the OCILY3 cell line. Survival analysis showed that high NINL expression was a risk factor for overall survival (OS) and disease-specific survival (DSS) within older patients and those with advanced-stage cancer. GSEA results showed that NINL may be involved in neutrophil-mediated immunity and NF-κB signaling.ConclusionThis study identified a novel LncRNA00654-NINL regulatory axis in DLBCL, which could provide a favorable reference for exploring the possible mechanisms of disease progression.

Mechanism of circZNF609 targeting miR-153 to regulate the proliferation and apoptosis of diffuse large B-cell lymphoma

Objective: To investigate the molecular mechanism of circZNF609 targeting miR-153 to regulate the proliferation and apoptosis of diffuse large B-cell lymphoma. Methods: Fifty cases of lymphoma tissue from patients with diffuse large B-cell lymphoma who were diagnosed and treated in the First Affiliated Hospital of Zhengzhou University from July 2018 to December 2019 were collected. Thirty cases of normal lymph node tissues that were confirmed to be reactive hyperplasia by pathological diagnosis during the same period were selected as controls. Real time quantitative polymerase chain reaction (PCR) was used to detect the expression of circZNF609 in diffuse large B-cell lymphoma tissues and control hyperplasia lymph nodes. Diffuse large B-cell lymphoma OCI-LY19 cells were divided into control group (blank control), si-con group (transfected with siRNA control), si-ZNF609 group (transfected with circZNF609 siRNA), and si-ZNF609+ Anti-NC group (co-transfected with circZNF609 siRNA and inhibitor control) and si-ZNF609+ Anti-miR-153 group (co-transfected with circZNF609 siRNA and miR-153 inhibitor). Cell counting kit-8 (CCK-8) was used to detected proliferation, flow cytometry was used to detect cell cycle and apoptosis. Western blot was used to detect the protein expressions of C-caspase-3, cyclin D1, p21. The luciferase reporter system was used to identifie the relationship between circZNF609 and miR-153. Results: The expression level of circZNF609 in diffuse large B-cell lymphoma tissue was (1.44±0.22), higher than (0.37±0.14) in the control tissues (P<0.001). The cell survival rate of the si-ZNF609 group was (51.74±6.39)%, lower than (100.00±10.23)% of the control group and the (99.64±11.67)% of the si-con group (P<0.001). The proportion of cells in the G(0)/G(1) phase was (63.25±4.11)%, higher than (48.62±4.32)% of the control group and (47.12±3.20)% of the si-con group (P<0.001), the apoptosis rate was (13.36±1.42)%, higher than (3.65±0.47)% of the control group and (3.84±0.62)% of the si-con group (P<0.05). The expression levels of C-caspase-3 and p21 protein were (0.85±0.09) and (0.90±0.08), higher than (0.38±0.04) and (0.65±0.07) in the control group and (0.39±0.05) and (0.66±0.05) in the si-con group (P<0.001). The expression level of cyclin D1 protein was (0.40±0.03), lower than (0.52±0.06) of the control group and (0.53±0.04) of the si-con group (all P<0.001). CircZNF609 and miR-153 are mutually targeted. The cell survival rate of the si-ZNF609+ Anti-miR-153 group was (169.92±13.25)%, higher than (100.00±9.68)% of the si-ZNF609+ Anti-NC group (P<0.001), the ratio of cells in G(0)/G(1) phase and apoptosis rate were (52.01±3.62)% and (8.20±0.87)%, respectively, lower than (64.51±5.17)% and (14.03±1.17)% in the si-ZNF609+ Anti-NC group (P<0.001). The protein expression levels of C-caspase-3 and p21 were (0.42±0.06) and (0.52±0.06), lower than (0.80±0.07) and (0.92±0.10) of the si-ZNF609+ Anti-NC group (P<0.001). The protein expression level of cyclin D1 was (0.68±0.07), higher than (0.39±0.04) in the si-ZNF609+ Anti-NC group (P<0.001). Conclusion: Down-regulation of circZNF609 inhibits the proliferation of diffuse large B-cell lymphoma OCI-LY19 cells and induces apoptosis by targeting miR-153.

CDK12 activates MYC to repress miR-28-5p/EZH2 and amplifies tonic BCR signaling to promote the development of diffuse large B-cell lymphoma.

Cyclin-dependent kinase 12 (CDK12) is a transcription-associated kinase that participates in various cellular processes. However, its regulatory role in the progression of diffuse large B-cell lymphoma (DLBCL), which is the most prevalent subtype of non-Hodgkin lymphoma (NHL), is still elusive and controversial.The expression of CDK12 was detected by immunohistochemistry (IHC), RT-qPCR was performed to detect miR-28-5p expression of OCI-LY3 and SU-DHL-4 cells. MTT and soft agarose colony formation assays were used to detect cell proliferation. The cell apoptosis was determined by flow cytometry. The protein expressions changes of MYC, EZH2 and the biomarkers of BCR signaling were also detected. A subcutaneous transplantation tumor model of OCI-LY3 cells in nude mice was established to evaluate anticarcinogenic activities of CDK12 knockdown. Elevated expression of CDK12 was observed while miR-28-5p was downregulated in DLBCL tissues. CDK12 knockdown or miR-28-5p overexpression could inhibit proliferation and promote apoptosis of DLBCL cells. miR-28-5p inhibition could reverse the effect of CDK12 knockdown on proliferation and apoptosis of DLBCL cells. In addition, CDK12 knockdown could inhibit DLBCL tumor growth in the mice model. CDK12 activated MYC to repress miR-28-5p/EZH2 and amplified tonic BCR signaling to promote the development of DLBCL, which might provide potential therapeutic targets for future therapeutic intervention in DLBCL.