BJAB Cells Research Articles

Targeting DLBCL by mutation-specific disruption of cancer-driving oncogenes.

Diffuse large B cell lymphomas (DLBCL) are highly aggressive tumors. Their genetic complexity and heterogeneity have hampered the development of novel approaches for precision medicine. Our study aimed to develop a personalized therapy for DLBCL by utilizing the CRISPR/Cas system to induce knockouts (KO) of driver genes, thereby causing cancer cell death while minimizing side effects. We focused on OCI-LY3 cells, modeling DLBCL, and compared them with BJAB cells as controls. Analysis of whole exome sequencing revealed significant mutations in genes like PAX5, CD79B, and MYC in OCI-LY3 cells. CRISPR/Cas9-mediated KO of these genes resulted in reduced cancer cell viability. Subsequent single and dual gRNA targeting of PAX5 mutations inhibited proliferation specifically in OCI-LY3 cells. Moreover, dual gRNA targeting of PAX5 and MYC induced chromosomal rearrangements, reducing cell proliferation substantially. However, targeting single intronic mutations did not affect cell viability, highlighting the importance of disrupting protein function. Targeting multiple mutations simultaneously addresses intra-tumoral heterogeneity, and the transient delivery of CRISPR/Cas9 allows for permanent gene disruption. While challenges such as incomplete editing efficiency and delivery limitations exist, further optimization may enhance therapeutic efficacy. Overall, our findings demonstrate the efficacy of CRISPR/Cas9 in targeting oncogenic mutations, opening avenues for precision medicine in DLBCL treatment.

EXPRESSION PATTERNS OF CD90, CD117, CD123, AND CD147 IN CONTINUOUS CELL LINES OF HUMAN MATURE B-CELL TUMORS

Summary. Aim: to analyze expression patterns of CD90, CD117, CD123, and CD147 in vitro in different continuous lines of human mature B-cell neoplasms. Objects and methods: expression patterns of the surface antigens on cells of the continuous lines of Burkitt lymphoma (Namalwa and BJAB), Hodgkin lymphoma (КМ-Н2), and follicular lymphoma (Car-1) were analyzed by flow cytometry with monoclonal antibodies against CD90, CD117, CD123, and CD147. Results: BJAB and Namalwa cells did not express CD90 and CD123, while a small fraction of cells (less than 2%) was CD117-positive. КМ-Н2 and Сar-1 cells did not express any of three above mentioned antigens. The percentage of CD147-positive cells in continuous lines of human mature B-cell neoplasms amounted to 80–100%. Conclusions: CD117 expression in cell populations of Burkitt lymphoma continuous cell lines is rather rare. CD90 and CD123 are not expressed in all cell lines of human mature B-cell neoplasms under study. At the same time, ubiquitous pattern of CD147 has been evident.

An HPK1 inhibitor enhanced the tumour response to anti-PD-1 immunotherapy in non-Hodgkin's lymphoma.

Anti-PD-1 immunotherapy has been widely applied in patients with some types of lymphoma. Classical Hodgkin's lymphoma (cHL) is highly sensitive to immunotherapy, but non-Hodgkin's lymphoma (NHL) does not show a good response. Studies have indicated that haematopoietic progenitor kinase 1 (HPK1) suppresses T cells and reduces antitumour immunity. Therefore, HPK1 inhibitors may restore and elicit antitumour immune responses and are promising candidate drug targets for cancer immunotherapy. We first explored the Gene Expression Profile Interactive Analysis (GEPIA) database and predicted that HPK1 expression was increased in diffuse large B-cell lymphoma (DLBCL) and associated with Nod-like receptor protein 3 (NLRP3) expression. We investigated whether an HPK1 inhibitor could enhance the tumour response to anti-PD-1 immunotherapy in NHL and the association between HPK1 and NLRP3 expression. Employing shHPK1 and an inhibitor, we demonstrated that the HPK1 inhibitor increased anti-PD-1-mediated T-cell cytotoxicity in BJAB and WSU-DLCL2 cells cocultured with peripheral blood mononuclear cells (PBMCs). HPK1 inhibitor treatment increased PD-1, PD-L1, Bax, p53 and NK-kB expression but decreased NLRP3 expression, indicating that the HPK1 inhibitor promoted apoptosis and blocked the NLRP3 inflammasome pathway to affect anti-PD-1-mediated T-cell cytotoxicity. Moreover, the HPK1 inhibitor enhanced the efficiency of anti-PD-1 immunotherapy in vivo in a zebrafish xenograft model of NHL. In summary, this study provides evidence that an HPK1 inhibitor enhanced the tumour response to anti-PD-1 immunotherapy in NHL by promoting apoptosis and blocking the NLRP3 pathway. These findings provide a potential therapeutic option for NHL combining HPK1 inhibitor treatment and anti-PD-1 immunotherapy.

Correlations between preclinical BJAB assay ranking of antisense drugs and clinical trial adverse events.

This analysis sought to assess the clinical predictivity of an in vitro assay which utilized the human B-lymphoma BJAB cell line, for identification of antisense oligonucleotides (ASOs) with the potential to elicit innate immune activation in humans. Adverse events (AEs) from clinical trial data were analyzed based on prior clinical knowledge and network analysis of the clinical data to identify correlations with the BJAB assay. Clinically evaluated ASOs were ranked by the BJAB assay's mean log-fold increase in TNF expression levels. Flu-like reactions (FLRs) and injection site reactions (ISRs), were chosen as AEs of interest, along with those Medical Dictionary for Regulatory Activities preferred terms identified using AE network analysis. Fifteen different 2'-O-methoxyethyl (2'MOE) modified ASOs were ranked by the incidence of each AE group in the integrated safety data from 35 clinical trials. ISRs are considered to be local to the injection site, whereas FLRs are reflected by systemic constitutional symptoms. The correlations identified in this analysis of integrated clinical data provide evidence that the ASO sequences selected by the BJAB assay have a lower likelihood of causing systemic inflammatory AEs associated with FLRs, but not ISRs.

Role of estrogen receptor signaling pathway-related genes in diffuse large B-cell lymphoma and identification of key targets via integrated bioinformatics analysis and experimental validation.

Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous malignancy. Epidemiologically, the incidence of DLBCL is higher in men, and the female sex is a favorable prognostic factor, which can be explained by estrogen. This study aimed to explore the potential targets of the estrogen receptor (ER) signaling pathway and provide a meaningful way to treat DLBCL patients. Datasets were obtained from the Gene Expression Omnibus (GEO) to identify differentially expressed genes (DEGs). Representative gene sets estrogen receptor pathways, and growth regulatory pathways were identified based on Gene Set Enrichment Analysis (GSEA) analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for function and pathway analysis. STRING and Cytoscape were used to construct the interaction network, and the MCODE plug-in performed the module analysis. GEPIA, TCGA, and LOGpc databases were used for expression and predictive analysis. The Human Protein Atlas (HPA) database was used to analyze the protein expression levels, cBioPortal was used to explore genetic alterations, and ROC analysis and prognostic assessment were used to predict the diagnostic value of genes. Finally, BJAB cells were treated with ER inhibitor fulvestrant and specific shRNA, and the expression of hub genes was verified by RT-qPCR. We identified 81 overlapping DEGs and CDC6, CDC20, KIF20A, STIL, and TOP2A as novel biomarkers affecting the prognosis of DLBCL. In addition, the STAT and KRAS pathways are considered potential growth regulatory pathways. These results hold promise for new avenues for the treatment of DLBCL patients.

Inflammatory Non-CpG Antisense Oligonucleotides Are Signaling Through TLR9 in Human Burkitt Lymphoma B Bjab Cells.

Nucleic acid-based phosphorothioate containing antisense oligonucleotides (PS-ASOs) have the potential to activate cellular innate immune responses, and the level of activation can vary quite dramatically with sequence. Minimizing the degree of proinflammatory effect is one of the main selection criteria for compounds intended to move into clinical trials. While a recently developed human peripheral blood mononuclear cell (hPBMC)-based assay showed excellent ability to detect innate immune active PS-ASOs, which can then be discarded from the developmental process, this assay is highly resource intensive and easily affected by subject variability. This compelled us to develop a more convenient high-throughput assay. In this study, we describe a new in vitro assay, utilizing a cultured human Bjab cell line, which was developed and validated to identify PS-ASOs that may cause innate immune activation. The assay was calibrated to replicate results from the hPBMC assay. The Bjab assay was designed to be high throughput and more convenient by using RT-qPCR readout of mRNA of the chemokine Ccl22. The Bjab assay was also shown to be highly reproducible and to provide a large dynamic range in determining the immune potential of PS-ASOs through comparison to known benchmark PS-ASO controls that were previously shown to be safe or inflammatory in clinical trials. In addition, we demonstrate that Bjab cells can be used to provide mechanistic information on PS-ASO TLR9-dependent innate immune activation.

The gallium complex KP46 sensitizes resistant leukemia cells and overcomes Bcl-2-induced multidrug resistance in lymphoma cells via upregulation of Harakiri and downregulation of XIAP in vitro

Tris-(8-quinolinolato)gallium(III) (KP46, AP-002) is an orally administered investigational anticancer and bone-protective drug currently being evaluated in patients with advanced solid tumors with bone involvement. Despite the clinical efficacy of other gallium compounds in non-Hodgkin’s lymphoma, effects of KP46 in hematological tumor settings have not been studied systematically before. We report here intriguing activities in various human cell lines, including such with multidrug resistance (MDR): In Nalm-6 lymphoblastic leukemia cell sublines, KP46 was capable of overcoming P-gp-related as well as P-gp-unrelated MDR. Apoptosis induction by KP46 was unaffected by bcl2-mediated vincristine-induced MDR in a BJAB lymphoma cell subline and even enhanced in a K562 leukemia subline with daunorubicin-induced MDR, which could be re-sensitized to daunorubicin by KP46. As the latter resistance is associated with lowered Harakiri (HRK) protein levels, a modulating effect of KP46 on HRK expression is suggested. This is consistent with the significant high upregulation of HRK on RNA and protein levels observed in KP46-treated parental BJAB cells according to qPCR and Western blot analysis, respectively. Furthermore, KP46 significantly reduces the protein level of X-linked inhibitor of apoptosis (XIAP) in BJAB cells, the most potent known inhibitor of apoptosis. Overall, these results indicate both a higher potential of HRK and XIAP as cellular targets for cancer therapy and a broader therapeutic potential of KP46 than hitherto envisaged.

Mechanism of LncHOTAIR Regulating Proliferation, Apoptosis, and Autophagy of Lymphoma Cells through hsa-miR-6511b-5p/ATG7 Axis.

Objective To explore the role of LncHOTAIR in apoptosis and autophagy in lymphoma. Methods The interaction between LncHOTAIR and miR-6511b-5p, as well as between miR-6511b-5p and ATG7, was verified by a dual luciferase assay. LncHOTAIR overexpression lentivirus was transducted and siATG7s were transfected into Raji and BJAB lymphoma cells, and the efficiency was verified by qPCR. Lymphocyte proliferation was detected by the cell counting kit-8 (CCK8) test, and autophagy was detected by transmission electron microscopy. The protein expressions of ULK1, Beclin1, ATG7, LC3, Bax, cleaved-caspase 3, and Bcl-2 were detected using Western blots. Results There was a targeting relationship between LncHOTAIR and miR-6511b-5p and between miR-6511b-5p and ATG7. LncHOTAIR overexpression promoted the proliferation and autophagy of Raji and BJAB cells, significantly upregulated ATG7, Beclin1, ULK1, Bcl-2, and LC3-II/LC3-I levels, and downregulated Bax and cleaved-caspase3 levels. siATG7 significantly inhibited the proliferation and autophagy of Raji and BJAB cells and promoted their apoptosis. Conclusion LncHOTAIR/hsa-miR-6511b-5p/ATG7 could regulate the proliferation, apoptosis, and autophagy of Raji and BJAB lymphoma cells.

Abstract A07: BTM 3566, a novel activator of the mitochondrial stress response promotes robust therapeutic responses in vitro and in vivo in diffuse large B-cell lymphoma

Abstract Relapsed/refractory diffuse large B-cell lymphomas (r/r-DLBCL) are a therapeutic challenge as they are highly heterogeneous both clinically and molecularly, which imposes a pressing need to develop novel therapies to improve outcomes in patients independently of the molecular subtype. We describe here BTM-3566, a first-in-class, orally active compound with activity against DLBCL. BTM-3566 induces apoptosis and complete cell killing in DLBCL lines a with an IC50 of ~200 – 500 nM. Responsive DLBCL cell lines include ABC, GCB, and double-hit and triple-hit lymphoma type. Pharmacokinetic studies in mice showed suitability for once daily dosing, with > 50% oral bioavailability and ~6 hours of serum half-life. In xenograft models using the double-hit DLBCL line SU-DHL-10, BTM-3566 treatment resulted in complete response in all tumor-bearing animals and durable remission in 50% of animals with no tumor growth occurring for 2 weeks after dose cessation. Expansion studies into human DLBCL PDX models harboring a range of high-risk genomic alterations demonstrated response in 100% of the lines with complete response in 6 of 9 PDX models tested. Dose scheduling studies indicate that 7 days of continuous dosing is sufficient to induce tumor regression and maintenance of a complete response. The therapeutic activity of BTM-3566 is related to a novel effect on mitochondrial proteostasis and a robust induction of the ATF4 ISR. Of the four eIF2α -kinases in the human genome we determined that HRI was uniquely required for BTM-3566 activity in DLBCL. HRI is activated by mitochondrial-related stress resulting in activation of the mitochondrial protease OMA1. Molecular analysis revealed that BTM-3566 activates OMA1, leading to induction of the ATF4 ISR pathway and apoptosis in DLBCL cell lines. Deletion of OMA1 reduces the ability of BTM-3566’s to induce apoptosis in DBLCL. Substrates of OMA1 include the dynamin OPA1 and DELE1, a protein recently shown to signal mitochondrial dysfunction through activation of HRI kinase and ATF4. Transfection of BJAB cells with a cleavage resistant OPA1 mutant has no effect on BTM-3566 induced apoptosis. In contrast, DELE1 KO suppresses BTM-3566 mediated apoptosis. BTM-3566 activates OMA1 without acting as a classical mitochondrial toxin. Instead, BTM-3566 induces OMA1 activity through a novel mechanism regulated by the mitochondrial protein FAM210B. FAM210B expression is negatively correlated with response to BTM-3566, and overexpression of FAM210B blocks OMA1 activation and causes complete resistance to BTM-3566 induced apoptosis. Taken together, these data support a novel antitumor mechanism in DLBCL, where BTM3566 induces mitochondrial stress, activating the OMA1-DELE1-HRI-eIF2a-ATF4 pathway leading to apoptosis and tumor regression. An Investigational New Drug application for BTM3566 in B-cell malignancies will be submitted in Q2 2022 with initiation of first in human clinical trials planned for fall 2022. Citation Format: Adrian Schwarzer, Matheus Oliveira, Marc-Jens Kleppa, Andy Anantha, Alan Cooper, Mark Hannink, Todd Hembrough, Jedd Levine, Michael Luther, Michael Stocum, Linsey Stiles, Marc Liesa-Roig, Matthew Kostura. BTM 3566, a novel activator of the mitochondrial stress response promotes robust therapeutic responses in vitro and in vivo 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 A07.

Abstract 3784: BTM-3655 co-opts mitochondrial quality control pathways to induce apoptosis, and complete tumor regression in DLBCL cell lines, xenografts and PDX models

Abstract Cellular integrated stress response (ISR) pathways are induced by multiple triggers - viral RNA, nutrient deprivation, protein misfolding and disruption of mitochondrial homeostasis -that lead to phosphorylation of eIF2α, blockade of translation and activation of transcription factor ATF4 allowing the cell to recover or become apoptotic. These pathways are emerging as important targets in oncology, and here we demonstrate that mitochondrial homeostatic pathways can be co-opted to induce apoptosis in tumor cells and complete tumor regression in xenograft and PDX models. Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous tumor, for which novel therapies are needed. We recently described BTM-3566, a small molecule which induces apoptosis in DLBCL cell lines, including double- and triple-hit subtypes. In xenograft models BTM-3566 induces complete and durable regression in all tumor-bearing animals, importantly, no regrowth is seen through 21 days post therapy. In an expanded panel of high-risk human DLBCL PDX models BTM-3566 demonstrated a 100% response rate, and complete tumor regression in 6 of 8 PDX models. Molecular analysis revealed that BTM-3566 activates the mitochondrial protease OMA1, leading to activation of HRI kinase, phosphorylation of the eIF2a -ATF4 pathway and apoptosis in DLBCL cell lines. CRISPR-Cas9 depletion of OMA1 eliminates BTM-3566’s apoptotic activity. Substrates of OMA1 include dynamin, OPA1 and DELE1, a protein of unknown function but which has recently been shown to act as a sensor for mitochondrial dysfunction and signals through HRI kinase and ATF4. Transfection of BJAB cells with a cleavage resistant OPA1 mutant has no effect on BTM-3566 induced apoptosis. In contrast, KO of DELE1 suppresses BTM-3566 mediated apoptosis. Interestingly, knockout of the ATF4 gene has a partial effect, delaying onset of apoptosis. BTM-3566 activates OMA1 in a manner unrelated to changes in mitochondrial ATP synthesis, reactive oxygen species generation or electron transport chain inhibition. Instead, BTM-3566 induces OMA1 activity through a novel mechanism regulated by the mitochondrial protein FAM210B. FAM210B expression is negatively correlated with response to BTM-3566, and overexpression of FAM210B blocks OMA1 activation and causes complete resistance to BTM-3566 induced apoptosis. Thus, FAM210B expression is a strong predictor of sensitivity to BTM-3566 and reveals a novel mechanism of regulation of OMA1 activation. Taken together, these data support a novel antitumor mechanism in DLBCL, where BTM3566 induces mitochondrial stress, activating the OMA1-DELE1-HRI-eIF2a-ATF4 pathway leading to apoptosis and tumor regression. An Investigational New Drug application for BTM3566 in B-cell malignancies will be submitted by early Q1 2022 with initiation of first in human clinical trials the first half of 2022. Citation Format: Adrian Schwarzer, Matheus Oliveira, Marc-Jens Kleppa, Andy Anantha, Alan Cooper, Todd Hembrough, Jedd Levine, Michael Luther, Michael Stocum, Marc Liesa-Roig, Matt Kostura. BTM-3655 co-opts mitochondrial quality control pathways to induce apoptosis, and complete tumor regression in DLBCL cell lines, xenografts and PDX models [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 3784.

B-nor-methylene Colchicinoid PT-100 Selectively Induces Apoptosis in Multidrug-Resistant Human Cancer Cells via an Intrinsic Pathway in a Caspase-Independent Manner.

Colchicine, the main active alkaloid from Colchicum autumnale L., is a potent tubulin binder and represents an interesting lead structure for the development of potential anticancer chemotherapeutics. We report on the synthesis and investigation of potentially reactive colchicinoids and their surprising biological activities. In particular, the previously undescribed colchicinoid PT-100, a B-ring contracted 6-exo-methylene colchicinoid, exhibits extraordinarily high antiproliferative and apoptosis-inducing effects on various types of cancer cell lines like acute lymphoblastic leukemia (Nalm6), acute myeloid leukemia (HL-60), Burkitt-like lymphoma (BJAB), human melanoma (MelHO), and human breast adenocarcinoma (MCF7) cells at low nanomolar concentrations. Apoptosis induction proved to be especially high in multidrug-resistant Nalm6-derived cancer cell lines, while healthy human leukocytes and hepatocytes were not affected by the concentration range studied. Furthermore, caspase-independent initiation of apoptosis via an intrinsic pathway was observed. PT-100 also shows strong synergistic effects in combination with vincristine on BJAB and Nalm6 cells. Cocrystallization of PT-100 with tubulin dimers revealed its (noncovalent) binding to the colchicine-binding site of β-tubulin at the interface to the α-subunit. A pronounced effect of PT-100 on the cytoskeleton morphology was shown by fluorescence microscopy. While the reactivity of PT-100 as a weak Michael acceptor toward thiols was chemically proven, it remains unclear whether this contributes to the remarkable biological properties of this unusual colchicinoid.

MiR-155-regulated mTOR and Toll-like receptor 5 in gastric diffuse large B-cell lymphoma.

BackgroundGastric diffuse large B‐cell lymphoma (DLBCL) is often associated with Helicobacter pylori (H. pylori) infection. Those in the early stage could be treated with H. pylori eradication therapy, and are classified into a sensitive group and a resistant group.MethodsGenome‐wide miRNA and miRNA expression profiles were obtained from biopsy specimens of gastric DLBCL. MiRNAs and their targets as predictors of responses to H. pylori eradication therapy were identified through differential expression and pathway enrichment analysis, and further confirmed with transfection experiments in lymphoma cell lines of B‐cell origin.ResultsGenome‐wide miRNA and mRNA profiles showed miR‐200 was associated with the sensitive group, and that the resistant group had higher levels of miR‐155 and lower levels of DEPTOR (an inhibitor of mTOR) than the sensitive group. BJAB cells transfected with miR‐155 also had lower DEPTOR and higher mTOR levels. Therefore, miR‐155‐mediated inhibition of DEPTOR with secondary activation of mTOR was a potential marker for resistance to H. pylori eradication therapy. In contrast, pathway enrichment analysis showed that Toll‐like receptor 5 (TLR5), the receptor for bacterial flagellin, was a potential marker for sensitivity to H. pylori eradication therapy. In an independent series, stronger expression of pS6K1 (a direct target of mTOR) was associated with the resistant group and morphologic evidence of active gastritis was associated with the sensitive group.ConclusionsThese findings showed that activation of the miR‐155‐DEPTOR pathway is a marker for resistance to H. pylori eradication therapy, and that histological evaluation of active gastritis might be used as a surrogate marker to predict responses to H. pylori eradication therapy in gastric DLBCL.

KP772 overcomes multiple drug resistance in malignant lymphoma and leukemia cells in vitro by inducing Bcl-2-independent apoptosis and upregulation of Harakiri

Despite high cure rates in pediatric patients with acute leukemia, development of resistance limits the efficacy of antileukemic therapy. Tris(1,10-phenanthroline)tris(thiocyanato-κN)lanthanum(III) (KP772) is an experimental antineoplastic agent to which multidrug-resistant cell models have shown hypersensitivity. Antiproliferative and apoptotic activities of KP772 were tested in leukemia, lymphoma and solid tumor cell lines as well as primary leukemia cells (isolated from the bone marrow of a child with acute myeloid leukemia (AML). The ability to overcome drug resistances was investigated in doxorubicin- and vincristine-resistant cell lines. Real-time PCR was used to gain insight into the mechanism of apoptosis induction. KP772 inhibited proliferation and induced apoptosis in various leukemia and lymphoma cell lines in a concentration-dependent manner (LC50 = 1–2.5 µM). Primary AML cells were also sensitive to KP772, whereas daunorubicin showed no significant effect. KP772 induces apoptosis independently of Bcl-2, Smac, and the CD95 receptor and is also effective in caspase 3-deficient MCF7 cells, indicating that apoptosis is partly triggered independently of caspase 3. mRNA expression profiling revealed an upregulation of the BH3-only Bcl-2 protein Harakiri in the course of KP772-induced apoptosis. Remarkably, KP772 overcame drug resistance to doxorubicin and vincristine in vitro, and the apoptotic effect in resistant cells was even superior to that in non-resistant parental cells. In combination with vincristine, doxorubicin and cytarabine, synergistic effects were observed in BJAB cells. The cytotoxic potency in vitro/ex vivo and the remarkable ability to overcome multidrug resistance propose KP772 as a promising candidate drug for antileukemic therapy, especially of drug-refractory malignancies.Graphic abstract

Cisplatin Loaded Poly(L-glutamic acid)-g-Methoxy Polyethylene Glycol Complex Nanoparticles Combined with Gemcitabine Presents Improved Safety and Lasting Anti-Tumor Efficacy in a Murine Xenograft Model of Human Aggressive B Cell Lymphoma.

Cisplatin (CDDP) is a highly effective anti-tumor drug with a broad spectrum of activity. However, the clinical efficacy of CDDP-containing regimens is yet unsatisfactory due to the severe dose-related toxicity of CDDP. In a previous study, CDDP nanoparticles (L-CDDP) forms a complex as CDDP with poly(L-glutamic acid)-g-methoxy poly(ethylene glycol) with improved safety compared to CDDP. Herein, a murine xenograft model of human aggressive B cell lymphoma (BCL) was established to explore anti-lymphoma efficiency of L-CDDP combined with GEM. BJAB cells represent an aggressive BCL, which were utilized to explore the anti-proliferative effect, cell apoptosis via CCK-8 test and flow cytometry technology, respectively. Toxicity experiment and the maximum tolerated dose (MTD) test were conducted in Kunming mice. Tumor inhibition experiment was conducted at the dose of MTD in SCID beige mice-bearing lymphoma. In this study, the loading capacity and encapsulating efficiency of CDDP in the L-CDDP was 18.3% and 89.7%, respectively, and the hydrodynamic diameter of the prepared L-CDDP was 20.6 nm. The CCK-8 data indicated that the anti-proliferative activity of monodrug groups (GEM, CDDP, L-CDDP) was dose- and time-dependent in BJAB cells. The synergistic effects in anti-lymphoma were detected in these two groups (GEM+CDDP, GEM + L-CDDP). Compared to control group, the proportion of apoptotic cells in experimental groups in BJAB cells was significantly higher at 48 h. Toxicity assays revealed that GEM + L-CDDP regimen had low hematological toxicity, hepatotoxicity, and nephrotoxicity. Tumor inhibition experiment demonstrated that GEM + L-CDDP group exhibited significant tumor-suppressing effects. Moreover, tumors continued to shrink in GEM + L-CDDP group, while these appeared to grow in the GEM + CDDP group. Finally, tumor necrosis was most prominent in the GEM + CDDP and GEM + L-CDDP groups, as assessed by hematoxylin-eosin staining. In conclusion, compared to CDDP, L-CDDP combined with GEM seriously induces BJAB cell apoptosis. Also, GEM + L-CDDP exhibits low hematotoxicity, hepatotoxicity, and nephrotoxicity. Importantly, GEM + L-CDDP presents lasting anti-lymphoma efficacy in a SCID beige mice-bearing lymphoma.

Triclosan induces apoptosis in Burkitt lymphoma-derived BJAB cells through caspase and JNK/MAPK pathways.

Burkitt's lymphoma (BL) is the fastest growing human tumor. Current treatment consists of a multiagent regimen of cytotoxic drugs with serious side effjects including tumor lysis, cardiotoxicity, hepatic impairment, neuropathy, myelosuppression, increased susceptibility to malignancy, and death. Furthermore, therapeutic interventions in areas of BL prevalence are not as feasible as in high-income countries. Therefore, there exists an urgent need to identify new therapies with a safer profile and improved accessibility. Triclosan (TCS), an antimicrobial used in personal care products and surgical scrubs, has gained considerable interest as an antitumor agent due to its interference with fatty acid synthesis. Here, we investigate the antitumor properties and associated molecular mechanisms of TCS in BL-derived BJAB cells. Dose-dependent cell death was observed following treatment with 10-100µM TCS for 24h, which was associated with membrane phospholipid scrambling, compromised permeability, and cell shrinkage. TCS-induced cell death was accompanied by elevated intracellular calcium, perturbed redox balance, chromatin condensation, and DNA fragmentation. TCS upregulated Bad expression and downregulated that of Bcl2. Moreover, caspase and JNK MAPK signaling were required for the full apoptotic activity of TCS. In conclusion, this report identifies TCS as an antitumor agent and provides new insights into the molecular mechanisms governing TCS-induced apoptosis in BL cells.

R-Nanochop Incorporating a TFR1-Targeted Doxorubicin Nanocarrier Is Superior to R-CHOP in a PDX Model of Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) comprises a third of non-Hodgkin lymphoma (NHL), with incidence steadily rising in what is already the most common hematologic malignancy. The backbone of potentially curative frontline chemoimmunotherapy combinations like R-CHOP is doxorubicin (Dox), still considered the most active anti-lymphoma agent. Relapsed or refractory (rel/ref) disease after frontline treatment carries persistently poor prognosis, even with recent advances in immunotherapy. Development and implementation of new therapeutic strategies remain urgent priorities to address unmet clinical needs in both high-risk previously untreated and rel/ref patients. We have studied transferrin receptor (TFR1) expression as a marker of high-risk DLBCL revealing significantly worse outcomes following frontline therapy associated with high expression in diagnostic samples. TFR1 is therefore a rational target for treatments aimed specifically at high-risk DLBCL. We used third-generation carbon-nitride dot (CND) nanocarriers we have developed conjugated to Dox and holo-transferrin (TF) to develop a chemotherapeutic-nanocarrier (NanoDox) designed to deliver Dox to TFR1-expressing tumors while sparing non-malignant tissues. Dox's mechanism is primarily through nuclear DNA damage or induction of reactive oxygen species (ROS) H202. We treated DLBCL cell lines with NanoDox or Dox for 24-hours and observed induction of nuclear DNA damage marker γ-H2Ax at drastically lower doses (10 nM NanoDox vs. 100 nM Dox). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay results yielded similar findings in BJAB cells, as observed under γ-H2Ax analysis, with TUNEL induction at significantly lower NanoDox concentrations. Treatment with NanoDox did not generate significant changes in ROS H202 when compared to Dox at 24-hours. Dox is known for a strong cytotoxic profile that extends past its terminal half-life of 24-36 hours. Therefore, we treated BJAB, Farage and DHL4 cells for 24-hours with a range of NanoDox and Dox doses, followed by washout and tracked cell viability for 6 additional days. We found as low as 10 nM NanoDox induced rapid and irreversible cytotoxicity, an effect only seen at much higher doses of Dox. These results combined with fluorescent confocal microscopy studies confirmed NanoDox works primarily through rapid nuclear Dox accumulation, causing DNA damage and apoptosis. Building on initial NanoDox dosing studies in NOD Scid Gamma (NSG) mice, we constructed R-nanoCHOP as an alternate to R-CHOP by replacing Dox with NanoDox at a working dose (WD) of 33.0 mg/kg. We next engrafted patient-derived xenograft (PDX) tumors derived from a previously untreated patient with germinal center B-cell (GCB) subtype DLBCL. We randomized NSG animals to two groups at tumor engraftment and treated with 21-day cycles of R-nanoCHOP (n=10) or R-CHOP (n=11). The treatments controlled tumor volume (TV) similarly, but R-nanoCHOP significantly improved overall survival associated with greatly reduced toxicities to host animals, which tolerated an average of 2 additional cycles of R-nanoCHOP. Studies with murine cells in vitro, including A20 B-cell lymphoma cells, confirmed these effects were not due to species differences, with human holo-TF alone and as part of NanoDox binding mouse and human TFR1 with equal affinity. This work establishes proof of principle in DLBCL for targeting TFR1, a specific marker of clinically high-risk disease. CND nanocarriers provide a flexible platform to exploit this, with our initial studies enhancing the safety profile of Dox, which remains the most active frontline DLBCL agent more than 50 years after it entered use. We are now optimizing NanoDox by substituting the holo-TF with an anti-TFR1 single chain variable fragment (scFv), decreasing size of the overall nanoconjugate by >50%. We believe dosing optimization from this change and additional optimizations to Dox delivery intracellularly will permit investigational new drug (IND) studies of absorption, distribution, metabolism and excretion (ADME). In sum, we provide compelling clinically relevant evidence for targeting TFR1 in DLBCL as a new therapeutic approach. Disclosures No relevant conflicts of interest to declare.

Abstract PO-47: BTM-3566 induces complete tumor regression in diffuse large B-cell lymphoma: Regulation by the eIF2α kinase “heme-regulated inhibitor” and the mitochondrial protein FAM210B

Abstract BTM-3566 is a novel small-molecule compound with broad anticancer activity in hematologic and solid cancers, with exceptional activity against diffuse large B-cell lymphoma (DLBCL). The compound's mechanism of action (MoA) is dependent on activation of the ATF4 integrated stress response (ISR) by the eIF2 kinase heme-regulated inhibitor (EIF2AK1, HRI) resulting in translational repression, increased expression of ATF4, and apoptosis. To evaluate HRI's role in mediating the ISR and apoptosis by BTM-3566 in DLBCL, homozygous HRI knockout BJAB cells were generated. HRI gene deletion resulted in inhibition of BTM-3566 induction of ATF4 expression, reduction of cell growth inhibition, and abrogation of apoptosis. Expression of the antiapoptotic proteins Mcl1 and Bcl6 was reduced following treatment with BTM-3566 in wild-type BJAB cells but not in HRI KO BJAB cells. To further elucidate BTM-3566 MoA, an activity screen was performed across 407 hematologic and solid cancer cell lines. Expression of FAM210B, a mitochondrial membrane protein, emerged as a key predictor of response to BTM-3566. FAM210B expression was negatively correlated with response to compound; the cell lines most responsive to BTM-3566 were the B-cell malignancies, notably DLBCL, which have the lowest expression of FAM210B among all cancers. The relevance of FAM210B to BTM-3566 activity was confirmed by overexpression of FAM210B in the DLBCL cell line BJAB and the Burkitt lymphoma cell line RAMOS. Overexpression of FAM210B abrogated BTM-3566 antiproliferative activity in these cell lines. The specificity of BTM-3566 activity regulation by FAM210B was demonstrated by comparison to treatments known to activate the ATF4 ISR through eIF2α kinases; tunicamycin (PERK), amino acid deprivation (GCN2), ONC201, and bortezomib (HRI) were tested in FAM210B-overexpressing BJAB cells. FAM210B overexpression inhibited BTM-3566-mediated induction of ATF4 protein expression but had no effect on ATF4 induction by the other treatments. The antitumor activity of BTM-3566 in DLBCL was evaluated in patient-derived DLBCL xenograft (PDX) mouse models. Eight models (n=3/model) were tested, which included GCB and ABC subtypes, and as typical for DLBCL, all had low FAM210B expression. Treatment was initiated with BTM-3566 at 20 mg/kg po daily after tumor volume reached 200 mm3. Complete tumor regression was observed in 79% of the mice with no evidence of tumor progression during the 21-day treatment period. Treatment was well tolerated with only mild weight loss noted. In summary, BTM-3566 induces robust complete regressions in DLBCL PDX models via a unique MoA characterized by apoptosis mediated by HRI induction of the ATF4 ISR in an FAM210B-regulated manner. BTM-3566 has the potential to meaningfully address unmet clinical needs in DLBCL and other B-cell malignancies with low FAM210B expression. Citation Format: Matthew Kostura, Jedd Levine, Alan Cooper, Andy Anantha, Michael Stocum, Michael Luther. BTM-3566 induces complete tumor regression in diffuse large B-cell lymphoma: Regulation by the eIF2α kinase “heme-regulated inhibitor” and the mitochondrial protein FAM210B [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-47.

Abstract PO-48: Cytotoxic mechanism of a novel transferrin receptor-targeting chemotherapeutic nanocarrier for use in diffuse large B-cell lymphoma

Abstract Diffuse large B-cell lymphoma (DLBCL), the most common hematologic malignancy, is an aggressive form of non-Hodgkin lymphoma. Approximately 60% of DLBCL patients achieve long-term disease-free survival from frontline R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), but patients with relapsed or refractory disease have poor prognosis. Doxorubicin (Dox) remains the most active anti-lymphoma agent, but its use is limited by well-characterized toxicities, including potentially irreversible cardiac damage. Targeted and specific delivery of Dox to tumors to overcome these limitations has been an area of active research. Targeted delivery of Dox via the transferrin receptor (TFR1) has preclinical efficacy in solid malignancies but has yet to be explored in lymphoma. We have uncovered for the first time a significant association between TFR1 expression and poor prognosis in DLBCL. Carbon-nitride dots (CNDs) are third-generation nanocarriers with inherent photoluminescence, which are easily conjugated with a wide variety of substrates. We conjugated Dox and transferrin (TF), the ligand for TFR1, to CNDs to develop CND-Dox-TF (CDT). CDT is uniquely designed to enhance Dox delivery to TFR1-expressing DLBCL tumors while limiting effects on nonmalignant tissues. BJAB and Farage cell lines treated with CDT for 24 hours underwent apoptosis at a significantly lower concentration when compared to Dox (50 nM vs. 1000 nM). Overall, CDT was 1-2 Log10 more potent than Dox against DLBCL cell lines. Co-incubation with 250 uM of antagonist holo-transferrin significantly decreased BJAB and Farage sensitivity to CDT, confirming activity through TFR1. Confocal microscopy of BJAB cells incubated up to 24 hours with 250 nM Dox and 30 nM CDT reveled enhanced nuclear colocalization of Dox at a significantly lower concentration. Prior dose-finding experiments in non-tumor bearing mice identified a CDT safe working dose of 33.0 mg/kg containing 16% moles of Dox in comparison to Dox maximum-tolerated dose (MTD) 3.3 mg/kg. We engrafted 2 groups of 10 NSG mice with a Dox-sensitive high-TFR1 expressing patient-derived xenograft (PDX) tumor. Mice were dosed with CDT and Dox on day 0, 14, and 24. CDT demonstrated similar anti-lymphoma efficacy with reduced toxicity: CDT-treated mice displayed no significant decline in body weight, which is characteristic and was observed following each single-agent Dox treatment. Histology analyses revealed little to no obvious damage to CDT-treated nonmalignant tissues, including myocardium. With a working dose of CDT identified, we now have under way clinically relevant R-CHOP vs. R-nanoCHOP (CDT substituted for Dox) assessments in our PDX mouse model. Capitalizing on the unique design, we expect to observe improved anti-lymphoma efficacy, with decreased toxicities and an improvement in overall survival. In sum, we provide mechanistic insight for novel DLBCL nano-chemotherapy and illustrate preclinical efficacy for a promising new therapeutic approach. Citation Format: Artavazd Arumov, Piumi Y. Liyanage, Asaad Trabolsi, Evan R. Roberts, Braulio Ferreira, Daniel Bilbao, Roger M. LeBlanc, Jonathan H. Schatz. Cytotoxic mechanism of a novel transferrin receptor-targeting chemotherapeutic nanocarrier for use in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-48.

Exogenous Expression of WNT7A in Leukemia-Derived Cell Lines Induces Resistance to Chemotherapeutic Agents.

Dysregulations of the WNT pathway are implicated in the malignant transformation of different types of neoplasia. WNT7A is expressed in normal peripheral lymphocytes, but is decreased in the tumoral counterpart. Furthermore, the treatment of leukemic cells with recombinant WNT7A decreases proliferation, suggesting its possible use as a therapeutic biomolecule. This study aimed to evaluate the concomitant action of WNT7A and different chemotherapeutic agents over proliferation and cell death of leukemia/ lymphoma derived cell lines. Ectopic expression of WNT7A was induced in CEM and BJAB cell lines by using a lentiviral system. RNA expression was analyzed by microarrays and qPCR, and protein expression was determined by Western Blot. Cell proliferation was measured by cell counting, metabolic activity by WST-1 assay, cell death and DNA content by flow cytometry. WNT7A ectopic expression was shown to decrease cell proliferation, but the apoptosis rate of leukemic cells was not altered. Moreover, these cells acquired resistance to doxorubicin, vincristine and MG-132. Cell cycle analysis reveals a decrease in G1 and an increase in S and G2 phases with a further upregulation of senescence- associated genes. Microarray analysis reveals that most gene expression changes were related to cancer and metabolic associated pathways. All those changes appear to be independent of the WNT canonical pathway regulation. WNT7A negatively regulates cell proliferation in leukemic cell lines and promotes resistance to chemotherapeutic agents by inducing a senescence-like phenotype independently of the WNT canonical pathway.

WNT974 Inhibits Proliferation, Induces Apoptosis, and Enhances Chemosensitivity to Doxorubicin in Lymphoma Cells by Inhibiting Wnt/β-Catenin Signaling.

BackgroundUpregulation of the Wnt/β-catenin pathway has been demonstrated to promote tumor proliferation and chemoresistance in lymphoma. Our objective was to evaluate the effect of the Wnt/β-catenin pathway inhibitor WNT974 in lymphoma cells.Material/MethodsHuman lymphoma cell lines HUT-78 and BJAB were treated with or without 1 μM WNT974±0.15 μg/L doxorubicin (Dox). Cell viability and proliferation were evaluated by CCK-8 and colony formation assay. Expression of proliferating cell nuclear antigen (PCNA), KI67, and apoptotic-related proteins including Bcl-2, Bax, cleaved-caspase3, and cleaved-caspase9, together with Wnt pathway proteins Wnt, β-catenin, Axin2, and c-Myc, were detected by Western blot analysis. Flow cytometry was used to calculate the ratio of apoptotic cells.ResultsIn HUT-78 and BJAB cells, 1 μM WNT974 significantly reduced viability and colony formation. The expression of 2 markers of tumor cell proliferation, protein PCNA and KI67, was also reduced by WNT974. Treatment with 1 μM WNT974 for 48 h increased the rate of cell apoptosis, inhibited the expression of anti-apoptotic protein Bcl-2, and enhanced pro-apoptotic proteins Bax, cleaved-caspase3, and cleaved-caspase9 expression in both cell lines. After treatment with WNT974 plus Dox, cell viability was markedly decreased compared with Dox treatment alone. Mechanistically, WNT974 prevented the expression of Wnt, Axin2, β-catenin, and its target gene c-Myc.ConclusionsWNT974 effectively treats lymphoma by inhibiting cell proliferation, inducing cell apoptosis, and enhancing chemosensitivity to Dox, and these effects are dependent on blocking Wnt/β-catenin signaling.