Mouse Model Of B-cell Lymphoma Research Articles

Inhibition of SRC-3 as a potential therapeutic strategy for aggressive mantle cell lymphoma.

Mantle cell lymphoma (MCL) has a poor prognosis and high relapse rates despite current therapies, necessitating novel treatment regimens. Inhibition of SRC-3 show effectiveness in vivo and in vitro in other B cell lymphomas. Additionally, previous studies have shown that SRC-3 is highly expressed in the lymph nodes of B cell non-Hodgkin's lymphoma patients, suggesting SRC-3 may play a role in the progression of B cell lymphoma. This study aimed to investigate novel SRC-3 inhibitors, SI-10 and SI-12, in mantle cell lymphoma. The cytotoxic effects of SI-10 and SI-12 were evaluated in vitro and demonstrated dose-dependent cytotoxicity in a panel of MCL cell lines. The in vivo efficacy of SI-10 was confirmed in two ibrutinib-resistant models: an immunocompetent disseminated A20 mouse model of B-cell lymphoma and a human PDX model of MCL. Notably, SI-10 treatment also resulted in a significant extension of survival in vivo with low toxicity in both ibrutinib-resistant murine models. We have investigated SI-10 as a novel anti-lymphoma compound via the inhibition of SRC-3 activity. These findings indicate that targeting SRC-3 should be investigated in combination with current clinical therapeutics as a novel strategy to expand the therapeutic index and to improve lymphoma outcomes.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Up-regulation of the PI3K/AKT and RHO/RAC/PAK signalling pathways in CHK1 inhibitor resistant Eµ-Myc lymphoma cells.

The development of resistance and the activation of bypass pathway signalling represents a major problem for the clinical application of protein kinase inhibitors. While investigating the effect of either a c-Rel deletion or RelAT505A phosphosite knockin on the Eµ-Myc mouse model of B-cell lymphoma, we discovered that both NF-κB subunit mutations resulted in CHK1 inhibitor resistance, arising from either loss or alteration of CHK1 activity, respectively. However, since Eµ-Myc lymphomas depend on CHK1 activity to cope with high levels of DNA replication stress and consequent genomic instability, it was not clear how these mutant NF-κB subunit lymphomas were able to survive. To understand these survival mechanisms and to identify potential compensatory bypass signalling pathways in these lymphomas, we applied a multi-omics strategy. With c-Rel-/- Eµ-Myc lymphomas we observed high levels of Phosphatidyl-inositol 3-kinase (PI3K) and AKT pathway activation. Moreover, treatment with the PI3K inhibitor Pictilisib (GDC-0941) selectively inhibited the growth of reimplanted c-Rel-/- and RelAT505A, but not wild type (WT) Eµ-Myc lymphomas. We also observed up-regulation of a RHO/RAC pathway gene expression signature in both Eµ-Myc NF-κB subunit mutation models. Further investigation demonstrated activation of the RHO/RAC effector p21-activated kinase (PAK) 2. Here, the PAK inhibitor, PF-3758309 successfully overcame resistance of RelAT505A but not WT lymphomas. These findings demonstrate that up-regulation of multiple bypass pathways occurs in CHK1 inhibitor resistant Eµ-Myc lymphomas. Consequently, drugs targeting these pathways could potentially be used as either second line or combinatorial therapies to aid the successful clinical application of CHK1 inhibitors.

Polysialylated nanoinducer for precisely enhancing apoptosis and anti-tumor immune response in B-cell lymphoma

B-cell lymphoma is one of the most common types of lymphoma, and chemotherapy is still the current first-line treatment. However, due to the systemic side effects caused by chemotherapy drugs, traditional regimens have limitations and are difficult to achieve ideal efficacy. Recent studies have found that CD22 (also known as Siglec-2), as a specific marker of B-cells, is significantly up-regulated on B-cell lymphomas. Inspired by the specific recognition and binding of sialic acid residues by CD22, a polysialic acid (PSA)-modified PLGA nanocarrier (SAPC NP) designed to target B-cell lymphoma was fabricated. Mitoxantrone (MTO) was further loaded into SAPC NP through hydrophobic interactions to obtain polysialylated immunogenic cell death (ICD) nanoinducer (MTO@SAPC NP). Cellular experiments confirmed that MTO@SAPC NP could be specifically taken up by two types of CD22+ B lymphoma cells including Raji and Ramos cells, unlike the poor endocytic performance in other lymphocytes or macrophages. MTO@SAPC NP was determined to enhance the ICD and show better apoptotic effect on CD22+ cells. In the mouse model of B-cell lymphoma, MTO@SAPC NP significantly reduced the systemic side effects of MTO through lymphoma targeting, then achieved enhanced anti-tumor immune response, better tumor suppressive effect, and improved survival rate. Therefore, the polysialylated ICD nanoinducer provides a new strategy for precise therapy of B-cell lymphoma. Statement of significance• Polysialic acid functionalized nanocarrier (SAPC NP) was designed and prepared.• SAPC NP is specifically endocytosed by two CD22+ B lymphoma cells.• Mitoxantrone-loaded nanoinducer (MTO@SAPC NP) promote immunogenic cell death and anti-tumor immune response.• “Polysialylation” is a potential new approach for precision treatment of B-cell lymphoma.

89Zr-ImmunoPET Shows Therapeutic Efficacy of Anti-CD20-IFNα Fusion Protein in a Murine B-cell Lymphoma Model.

Antibody-mediated tumor delivery of cytokines can overcome limitations of systemic administration (toxicity, short half-lives). Previous work showed improved antitumor potency of anti-CD20-IFNα fusion proteins in preclinical mouse models of B-cell lymphoma. Although tumor targeting is mediated by the antibody part of the fusion protein, the cytokine component might strongly influence biodistribution and pharmacokinetics, as a result of its affinity, size, valency, and receptor distribution. Here, we used immunoPET to study the in vivo biodistribution and tumor targeting of the anti-CD20 rituximab-murine IFNα1 fusion protein (Rit-mIFNα) and compared it with the parental mAb (rituximab, Rit). Rit-mIFNα and Rit were radiolabeled with zirconium-89 (89Zr, t1/2 78.4 hours) and injected into C3H mice bearing syngeneic B-cell lymphomas (38C13-hCD20). Dynamic [(2 hours post injection (p.i.)] and static (4, 24, and 72 hours) PET scans were acquired. Ex vivo biodistribution was performed after the final scan. Both 89Zr-Rit-mIFNα and 89Zr-Rit specifically target hCD20-expressing B-cell lymphoma in vivo. 89Zr-Rit-mIFNα showed specific uptake in tumors (7.6 ± 1.0 %ID/g at 75 hours p.i.), which was significantly lower than 89Zr-Rit (38.4 ± 9.9 %ID/g, P < 0.0001). ImmunoPET studies also revealed differences in the biodistribution, 89Zr-Rit-mIFNα showed rapid blood clearance and high accumulation in the liver compared with 89Zr-Rit. Importantly, immunoPET clearly revealed a therapeutic effect of the single 89Zr-Rit-mIFNα dose, resulting in smaller tumors and fewer lymph node metastases compared with mice receiving 89Zr-Rit. Mice receiving 89Zr-Rit-mIFNα had enlarged spleens, suggesting that systemic immune activation contributes to therapeutic efficacy in addition to the direct antitumoral activity of IFNα. In conclusion, immunoPET allows the noninvasive tracking and quantification of the antibody-cytokine fusion protein and helps understand the in vivo behavior and therapeutic efficacy.

Allogeneic CAR Invariant Natural Killer T Cells Exert Potent Antitumor Effects through Host CD8 T-Cell Cross-Priming.

The development of allogeneic chimeric antigen receptor (CAR) T-cell therapies for off-the-shelf use is a major goal that faces two main immunologic challenges, namely the risk of graft-versus-host disease (GvHD) induction by the transferred cells and the rejection by the host immune system limiting their persistence. In this work we assessed the direct and indirect antitumor effect of allogeneic CAR-engineered invariant natural killer T (iNKT) cells, a cell population without GvHD-induction potential that displays immunomodulatory properties. After assessing murine CAR iNKT cells direct antitumor effects in vitro and in vivo, we employed an immunocompetent mouse model of B-cell lymphoma to assess the interaction between allogeneic CAR iNKT cells and endogenous immune cells. We demonstrate that allogeneic CAR iNKT cells exerted potent direct and indirect antitumor activity when administered across major MHC barriers by inducing tumor-specific antitumor immunity through host CD8 T-cell cross-priming. In addition to their known direct cytotoxic effect, allogeneic CAR iNKT cells induce host CD8 T-cell antitumor responses, resulting in a potent antitumor effect lasting longer than the physical persistence of the allogeneic cells. The utilization of off-the-shelf allogeneic CAR iNKT cells could meet significant unmet needs in the clinic.

Establishing Mouse Models of B-Cell Non-Hodgkin's Lymphoma

There are numerous types of B-cell lymphoma including Burkitt lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma/mucosa-associated lymphoid tissue lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma and other less frequent subtypes. However, the molecular pathogenesis of B-cell lymphoma remain largely unknown. Here we chose three different B-cell lymphoma subtype cell lines to establish mouse models, which could be useful to determine the efficacy and mechanisms of novel therapies.Firstly, we injected subcutaneously lymphoma cells into the right axillary region of BALB/c (nu/nu) nude mice. DOHH2 F0, Ly8 F0, Raji F0 appeared 7, 7, 21 days later. When tumors growed to 1.5cm at length, successful passage of mouse models was established by injecting cell suspension obtained from mincing and grinding tumors through a 70-μm cell strainer. The cell suspension was cultured at 37°C in a humidified atmosphere containing 5% CO2. The cells were cryopreserved in cryopreservation-medium (90% FBS, 10% DMSO), stored in liquid nitrogen, thawed again (with a viability of more than 70%) and successfully reconstituted. The above cell lines mouse models have been passaged to the 16th, 18th, 13th generation.For histological examination, an autopsy was performed on each passage of nude mice, and immunohistochmistry was use to determine the expression of surface markers. The tumor tissues from DOHH2 F0, DOHH2 F6, DOHH2 F12 were positive for CD20, cytoplasmic CD3, CD10, CD5, CD21, Ki67, CyclinD1, Bcl-2, Bcl-6. EBV was also detected in situ hybridization. Grade 3B Follicular lymphoma was identified in this B-cell lymphoma and was consistent with the primary origination of DOHH2. Immunohistochemical staining of Ly8 F0, Ly8 F6, Ly8 F12 showed the large atypical cells were positive for CD20, CD3, CD10, Bcl-2, mum-1, no reactivity to antibodies for bcl-6 and negative to EBER. Ki-67 values were graded as 30%. The pathologic expression in Raji F0, Raji F6, Raji F12 is like the following: CD20+/CD3+-/Ki67+-/CD10+/Bcl2+-/ Bcl6- /Mum1+/EBER++.Flow cytometry analysis was performed on cell suspensions cultured from each passage tumor tissues. DOHH2 F0, DOHH2 F6,DOHH2 F12 cells were CD19+/CD20-/CD10+/CD5-/CD38+/HLA-DR+/ CD22+ /CD23+/Ki-67+CD71+, and the kappa light chain was expressed monoclonally. Similar to DOHH2 cells, the serial Ly8 cells were CD19+ /CD20-/CD10+/CD5-/CD38+/HLA-DR+/CD22+/CD23+Ki-67+CD71+/EBER-, and the kappa light chain was expressed monoclonally. As Burkitt lymphoma, Raji and its serial cell clones were CD19+/CD20+/CD10+/CD5-/CD38+/HLA-DR+/CD22+/Ki-67+/CD71+/EBER++.In conclusions, we firstly and systematically established mouse models of B-cell lymphoma and take into passage successfully. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A New PET Probe for Imaging Chimeric Antigen Receptor T Cells.

HomeRadiology: Imaging CancerVol. 3, No. 2 PreviousNext Research HighlightsFree AccessA New PET Probe for Imaging Chimeric Antigen Receptor T CellsGary D. LukerGary D. LukerGary D. LukerPublished Online:Mar 26 2021https://doi.org/10.1148/rycan.2021219004MoreSectionsPDF ToolsImage ViewerAdd to favoritesCiteTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinked In Take-Away Points■ Major Focus: To investigate the feasibility of imaging chimeric antigen receptor (CAR) T cells with a zirconium 89–labeled antibody targeted to inducible T-cell costimulator (ICOS or CD278).■ Key Result: ImmunoPET imaging of ICOS enabled the visualization of activated CAR T cells without affecting persistence or function of these cells in a mouse model of B-cell lymphoma.■ Impact: ImmunoPET with antibody detection of the endogenous activation marker ICOS provides a potentially general approach to image any CAR T-cell therapy.CAR T cells are a type of cancer therapy based on engineering a patient’s own T cells with a receptor for a specific protein target on cancer cells. After expansion of the cells in a laboratory, a patient receives an infusion of the engineered CAR T cells. For efficacy, the cells must migrate to tumors, proliferate, as well as become and remain activated to kill target cancer cells. CAR T cells have shown impressive successes in some blood cancers, such as B-cell malignancies, but efficacy remains limited in solid tumors. A major challenge in understanding successes and failures of CAR T-cell therapy is the ability to monitor these cells in vivo. Current methods rely on measuring numbers of cells in the circulation, but this approach does not reliably reflect actual migration of CAR T cells into a tumor.Simonetta and colleagues analyzed human and mouse CAR T cells and identified ICOS as a promising marker of activation to an antitumor state. The team labeled an antibody to ICOS with zirconium 89, providing a probe for PET imaging of CAR T cells in a mouse model of B-cell lymphoma. PET imaging showed greater accumulation of the immunoPET probe to ICOS in mouse bone marrow sites of lymphoma. Tracer doses of the immunoPET antibody did not affect persistence or function of CAR T cells as cancer immunotherapeutics. While the current study investigated therapy in mice, the study suggests that immunoPET imaging of ICOS could become a clinically translatable, general approach to monitor cancer therapy with a wide range of CAR T cells.Highlighted ArticleSimonetta F, Alam IS, Lohmeyer JK, et al. Molecular imaging of chimeric antigen receptor T cells by ICOS-ImmunoPET. Clin Cancer Res 2021;27:1058–1068. doi: https://doi.org/10.1158/1078-0432.CCR-20-2770Highlighted ArticleSimonetta F, Alam IS, Lohmeyer JK, et al. Molecular imaging of chimeric antigen receptor T cells by ICOS-ImmunoPET. Clin Cancer Res 2021;27:1058–1068. doi: https://doi.org/10.1158/1078-0432.CCR-20-2770 Crossref, Medline, Google ScholarArticle HistoryPublished online: Mar 26 2021 FiguresReferencesRelatedDetailsRecommended Articles CD38-targeted Immuno-PET of Multiple Myeloma: From Xenograft Models to First-in-Human ImagingRadiology2020Volume: 295Issue: 3pp. 606-615Nanotechnology for Cancer Imaging: Advances, Challenges, and Clinical OpportunitiesRadiology: Imaging Cancer2021Volume: 3Issue: 318F FDG PET/CT and Novel Molecular Imaging for Directing Immunotherapy in CancerRadiology2022Volume: 304Issue: 2pp. 246-264Identification of HER2-Positive Metastases in Patients with HER2-Negative Primary Breast Cancer by Using HER2-targeted 89Zr-Pertuzumab PET/CTRadiology2020Volume: 296Issue: 2pp. 370-378Radiotheranostics in Cancer Diagnosis and ManagementRadiology2018Volume: 286Issue: 2pp. 388-400See More RSNA Education Exhibits Immune Effector Cell Therapy for Solid Tumors: Opportunities for Interventional RadiologyDigital Posters2019When Things Go Wrong, Characteristic PET CT Patterns of Triple and Double-Hit Lymphoma; A Pictorial ReviewDigital Posters2018Imaging Biomarkers in Targeted Therapies: From Quantitative Imaging to RadiomicsDigital Posters2019 RSNA Case Collection Benign bone marrow stimulation RSNA Case Collection2020Primary Calvarial Lymphoma RSNA Case Collection2021Primary Bone LymphomaRSNA Case Collection2020 Vol. 3, No. 2 Metrics Altmetric Score PDF download

Transcriptional Activation of MYC-Induced Genes by GCN5 Promotes B-cell Lymphomagenesis.

Overexpression of the MYC oncoprotein is an initiating step in the formation of several cancers. MYC frequently recruits chromatin-modifying complexes to DNA to amplify the expression of cancer-promoting genes, including those regulating cell cycle, proliferation, and metabolism, yet the roles of specific modifiers in different cancer types are not well defined. Here, we show that GCN5 is an essential coactivator of cell-cycle gene expression driven by MYC overexpression and that deletion of Gcn5 delays or abrogates tumorigenesis in the Eμ-Myc mouse model of B-cell lymphoma. Our results demonstrate that Gcn5 loss impacts both expression and downstream functions of Myc. SIGNIFICANCE: Our results provide important proof of principle for Gcn5 functions in formation and progression of Myc-driven cancers, suggesting that GCN5 may be a viable target for development of new cancer therapies.

AVM0703 as a novel stand alone or combination therapy for lymphoma.

42 Background: Chemotherapy remains a vital part of cancer treatment. There is an urgent need to reduce the toxicities associated with chemotherapy-based treatments especially in the pediatric and young adult population where the risk for chemo-induced secondary cancers is high. AVM0703 is a new treatment option that can be used as a stand-alone or in combination with chemo for relapsed/refractory lymphoma patients. AVM0703 is effective at lymphoablating while sparing red blood cells, platelets, and hematopoietic stem cells. AVM0703 is a novel patent-pending formulation of a repurposed drug without the toxic excipients unlike existing formulations. AVM0703 has anti-tumor activity in an immunocompetent mouse model of B-cell lymphoma (A20) as both a stand-alone treatment and in combinations with current therapeutics. Methods: Wild-type BALB/c mice were implanted with A20 tumor cells suspended in a 50% Matrigel mixture and implanted subcutaneously. Palpable tumors were visible within seven days. Mice were dosed with either high-dose AVM0703 alone, or in combination with intraperitoneal cyclophosphamide and fludarabine. Mice were then monitored for tumor growth and changes in body weight. Results: Mice dosed repeatedly with AVM0703 survived 20 days longer than mice dosed with placebo and displayed no significant toxicity or body weight loss. In a separate study, mice treated with 2 doses of Cy/Flu had a loss of tumors similar to 1 dose of AVM0703 + 1 dose of Cy/Flu. Additionally, when compared to standard treatments like CHOP, in the same A20 lymphoma model, the AVM0703 + Cy/Flu combination had better tumor efficacy than 2 cycles of CHOP. Unlike 2XCHOP treatment, which induced greater than 30% body weight loss and death of 18% of the mice due to drug toxicity, the AVM0703 + Cy/flu combination had no observed toxicity or body weight loss. Conclusions: AVM0703 can reduce the number of chemotherapy cycles without affecting efficacy and decrease the toxicities associated with chemo and will help to provide a better quality of life for patients.

Absorption, Distribution, and Binding Profile of ME-401, a Potent and Selective Oral Small-Molecule Inhibitor of Phosphatidylinositol 3-Kinase δ (PI3Kδ) in Animal and B-Cell Lymphoma Models.

BackgroundPhosphatidylinositol 3-kinase isoform δ (PI3Kδ) mediates multiple events in lymphocytes, including cell proliferation, survival, and motility. Inhibition of PI3Kδ, with downstream inhibitory effects on cell growth and survival, has been utilized to treat lymphoid malignancies. ME-401 is an oral, once-daily, selective PI3Kδ inhibitor with an optimized pharmacologic profile that is in clinical development for the treatment of B-cell malignancies.ObjectivesThis work examined aspects of the pharmacologic profile of ME-401 in preclinical models to investigate the basis of the clinical activity of ME-401 that may differentiate it from other currently approved PI3Kδ inhibitors.MethodsWe determined the ME-401 blood to plasma ratios, permeability, and purified enzyme-binding kinetics. The oral bioavailability and volume of distribution of ME-401 were evaluated in various species. ME-401 concentrations in plasma and tumor and brain tissues were evaluated following oral administration in an A20 syngeneic mouse model of B-cell lymphoma. Idelalisib was used as a reference compound for the measurement of purified enzyme-binding kinetics and concentrations in plasma and tumor in the A20 syngeneic mouse model.ResultsOral administration of ME-401 to dogs resulted in 79% bioavailability compared with intravenous administration. Allometric scaling from rodents, dogs, and nonhuman primates resulted in a predicted human volume of distribution at steady state of 10.75 L/kg. ME-401 was shown to distribute into the lymph in dogs and permeate into cells readily, with a human blood to plasma ratio of 1.4 and ~ 50% retention in the Caco-2 cell monolayer at 1 μM. The high binding affinity and low dissociation rate of ME-401 resulted in an equilibrium dissociation constant (KD) of 3.03 × 10−11 M. Oral administration of ME-401 in an A20 syngeneic mouse model resulted in tumor concentrations 20–30 times higher than plasma concentrations at 4 h after the last dose. By 24 h, the tumor levels had decreased approximately 30–50% compared with levels at 4 h while remaining significantly increased relative to plasma concentrations. ME-401 was also present in brain tissue at 4 and 24 h after the last dose. In comparison, the idelalisib dissociation rate was ~ 100 times higher, resulting in a KD of 1.11 × 10−9 M. Idelalisib tumor concentrations were only approximately three times higher than plasma concentrations at 4 h, while dropping below the limit of quantitation in both tumor and plasma by 24 h.ConclusionsThese data support the capacity of ME-401 to be orally absorbed, distribute to target tissues, enter and accumulate in target cells, and bind to the target with high affinity to exert its mechanism of action. These characteristics underlie the high clinical potency seen in B-cell malignancies that may differentiate ME-401 from other PI3Kδ inhibitors currently approved or in development.

Dino Is a DNA Damage-Induced Tumor Suppressing Long Noncoding RNA

While the CKDN1A locus is frequently suppressed in human cancers, the mechanism by which this finding contributes to tumor suppression has not been fully elucidated. We hypothesized that DINO, a novel DNA damage-induced long noncoding RNA encoded in the CDKN1A promoter, is essential to p53-dependent tumor suppression. We used a combination of in vivo mouse models, cell culture experiments and human tumor profiling to examine the contribution of DINO to p53-dependent tumor suppression. We crossed a Dino loss of function mouse to the E-mu-Myc model of B-cell lymphoma to examine p53-dependent, p21-independent tumor suppression. Rescue experiments to restore Dino function in Dino-/- lymphoma cells were performed to confirm its contribution to growth suppression. Finally, we analyzed methylation patterns of the DINO/CDKN1A locus in TCGA datasets to investigate the relationship of DINO inactivation and TP53 mutation in human cancers. We find that loss of Dino significantly accelerates tumorigenesis in the E-mu-Myc mouse model of B-cell lymphoma in a haplo-insufficient manner, while previous studies have shown that loss of Cdkn1a does not impact tumor development. Complementation with Dino suppresses growth of isolated Dino-/-, E-mu-Myc lymphoma cells and delays tumor development when re-engrafted into mice. We show that each of these effects requires an intact p53 signaling pathway. Human DINO and mouse Dino can each functionally restore growth suppression in cell culture, suggesting functional conservation through mammalian evolution. Finally, we find that DINO is hypermethylated in multiple types of human cancer and that DINO hypermethylation is correlated to DINO expression, but not CDNK1A expression. Hypermethylation of DINO is mutually exclusive with TP53 alteration in several specific human cancers. We provide direct evidence that the long noncoding RNA DINO is a tumor suppressor transcribed from the CDKN1A promoter. These findings provide insight into a mechanism by which human tumors inactivate p53-dependent functions and describe a novel mechanism of tumor suppression by a long noncoding RNA. We suggest that inactivation of DINO presents a potential biomarker of tumors with altered p53 function, and provides a future basis for targeted therapy and personalized treatment.

Abstract 2548: DNA damage response gene expression in CHK1 inhibitor responsive and resistant mouse models of MYC driven B-cell lymphoma

Abstract Enhanced tumor replication stress and genomic instability can lead to disruption of the DNA damage response, leading to tumor survival and dependence on essential checkpoint controls. Drugs to target intact pathways, such as CHK1 inhibitors, are therefore desirable and currently in clinical trials as potential future anticancer therapies. The underlying mechanisms underpinning the response and resistance to CHK1 inhibitor treatment are still unclear. We have demonstrated that NF-κB can regulate the response to oncogene induced DNA replication stress in vivo through regulation of CLSPN (Claspin) gene expression. Claspin is an essential adaptor protein required for the phosphorylation and activation of CHK1 by ATR. Using the Eµ-MYC mouse model of B-cell lymphoma we have found that mutation of NF-κB subunits (knockout of c-Rel or a T505A transactivation domain mutation of RelA) leads to earlier lymphoma onset that correlates with loss of Clspnexpression and inhibition of CHK1 activity. Moreover, while wild type Eµ-MYC lymphomas are highly responsive to CHK1 inhibitor treatment, by contrast Eµ-MYC NF-κB mutant cRel-/- or RelAT505A lymphomas are resistant. To understand more about how MYC driven lymphoma cells respond to CHK1 inhibitor treatment, we have investigated changes in the expression of genes encoding members of DNA damage response pathways. Wild type mice reimplanted with Eµ-MYC lymphoma cells were treated with the CHK1 inhibitor CCT244747 and harvested at multiple fixed timepoints. Quantitative PCR analysis of mRNAs encoding proteins in the ATR/CHK1 and ATM/CHK2 pathways was performed, with RPL13A used as reference gene. Interestingly, while dramatic changes were seen in wild type Eµ-MYC cells, these were not seen in CHK1 inhibitor resistant cRel-/- lymphomas. We observed an upregulation of the DNA damage and cell cycle responsive genes in the CHK1 inhibitor sensitive wild-type Eµ-MYC mice. This is consistent with with previously published in vitro studies and suggests an S phase cell cycle arrest in response to CHK1 inhibitor induced DNA damage. This response, however, is absent in the CHK1 inhibitor resistant Eµ-MYC NF-κB mutant lymphomas. It is hoped that by understanding these differences in gene expression that biomarkers of CHK1 inhibitor response can be identified to help stratify future clinical candidates. Citation Format: Nicola L. Hannaway, Jill E. Hunter, Alastair Greystoke, Neil D. Perkins. DNA damage response gene expression in CHK1 inhibitor responsive and resistant mouse models of MYC driven B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2548.

Traceless aptamer-mediated isolation of CD8+ T cells for chimeric antigen receptor T-cell therapy

Chimeric antigen receptor (CAR) T-cell therapies using defined product compositions require high-purity T-cell isolation systems that, unlike immunomagnetic positive enrichment, are inexpensive and leave no trace on the final cell product. Here, we show that DNA aptamers, generated with a modified cell-SELEX (systematic evolution of ligands by exponential enrichment) procedure to display low nanomolar affinity for the T-cell marker CD8, enable the traceless isolation of pure CD8+ T cells at low cost and high yield. Captured CD8+ T cells are released label-free by complementary oligonucleotides that undergo toehold-mediated strand displacement with the aptamer. We also show that CAR-T cells manufactured from these cells are comparable to antibody-isolated CAR-T cells in proliferation, phenotype, effector function and antitumor activity in a mouse model of B-cell lymphoma. By employing multiple aptamers and the corresponding complementary oligonucleotides, aptamer-mediated cell selection could enable the fully synthetic, sequential and traceless isolation of desired lymphocyte subsets from a single system.

Novel Combination Therapy Targeting rDNA Transcription and Histone Deacetylation Provides Effective Treatment for Multiple Myeloma, and Synergises in Bortezomib-Resistant MM

Background: Multiple myeloma (MM) requires combination drug therapies to delay acquired drug resistance and clinical relapse. We co-developed CX-5461, a highly-selective inhibitor of RNA polymerase I-mediated rDNA transcription(1), currently in phase I trials for relapsed haematological malignancies (Peter Mac). CX-5461 produces a targeted nucleolar DNA damage response (DDR), triggering both a p53-dependent and -independent nucleolar stress response and killing malignant cells while sparing normal cells(2,3). Single-agent CX-5461 provides an impressive survival benefit in mouse models of B-cell lymphoma, acute myeloid leukaemia and now MM(2,4,5). However, drug resistance eventually occurs, confirming the need for combination therapies.Aim: To test the efficacy of CX-5461 in combination with the histone deacetylase inhibitor panobinostat, (prioritised from a boutique high-throughput screen of anti-myeloma agents), with a focus on the setting of resistance to proteasome-inhibitors (PIs).Methods: We assessed the impact of CX-5461 and panobinostat on overall survival in mouse models of MM, then surveyed the effects on cellular response and molecular markers of DDR. We developed bortezomib-resistant cell lines and an in vivo model of bortezomib-resistance to test this combination in the setting of PI-resistance.Results: CX-5461 in combination with panobinostat provides a significant survival advantage in both the transplanted Vk*MYC and the 5T33/KaLwRij models, with minimal bone marrow toxicity.The combination showed increased anti-proliferative effects and cell death in vitro. Interestingly, experiments interrogating the downstream cellular response of this combination suggest that the mechanism(s) driving synergy are complex and cell context-dependent. Cell cycle analysis indicates that both CX-5461- and panobinostat-driven cell cycle effects, i.e. G2/M and G1/S arrest, respectively, are dominant in the combination setting in a cell line-dependent manner, suggesting that context-dependent factors such as p53 may influence the cellular response.Mechanistically, in both p53-wild type and -null cell lines we observe an increase in DDR signalling with single agent CX-5461, with only moderate further increase with the combination. Moreover, CX-5461-mediated MYC downregulation is not universally observed, with the combination promoting further downregulation only in some cell lines. Given the potential for affecting global transcription programs downstream of panobinostat, we are performing transcriptome analyses in the combination setting compared to single agent treatment.We have generated bortezomib-resistant cell lines, sequentially increasing drug exposure to establish populations growing at concentrations above the IC90 of the parental lines. The resistant 5T33 cells retain their resistance to bortezomib in vivo and we have demonstrated that CX-5461 remains effective in this model, significantly increasing survival. We are currently examining the combination of CX-5461 with panobinostat in this model of bortezomib-resistance, which will give critical information guiding patient selection for future clinical trials.Conclusion: The rDNA transcription inhibitor CX-5461 synergises in vitro and in vivo with panobinostat, and CX-5461 retains efficacy in the setting of bortezomib-resistant myeloma.ReferencesDrygin et al., Cancer Research 2011Bywater et al., Cancer Cell 2012Quin et al, Oncotarget, 2016Devlin et al., Cancer Discovery 2016Hein et al., Blood 2017 DisclosuresHarrison:Janssen-Cilag: Other: Scientific advisory board.

β-Adrenergic Signaling Impairs Antitumor CD8+ T-cell Responses to B-cell Lymphoma Immunotherapy.

β-Adrenergic receptor (βAR) signaling regulates many physiological processes, including immune system responses. There is growing evidence also for βAR-induced modulation of cancer growth and metastasis. In the Eμ-myc mouse model of B-cell lymphoma, we investigated the effects of chronically elevated βAR signaling on lymphoma progression and antitumor immunity, as well as the impact on cancer immunotherapy. Chronic treatment with the nonselective β-agonist isoprenaline promoted lymphoma development in a manner dependent on signaling within the hematopoietic compartment. βAR signaling significantly suppressed the proliferation, IFNγ production, and cytolytic killing capacity of antigen-specific CD8+ T cells. This inhibited CD8+ T-cell responses to immune modulating antibodies, including anti-PD-1 and anti-4-1BB, resulting in less effective control of lymphoma. The inhibitory effects on CD8+ T cells occurred independently of changes to DC function and included direct suppression of CD8+ T-cell stimulation. The suppressive effects of chronic βAR signaling on antitumor effector cells was selective to T cells, as it did not perturb the innate lymphocyte response to an experimental NKT cell-targeting vaccine, in a setting where innate immune control is dependent on NKT cell and NK cell activation. These findings demonstrate that chronic βAR signaling has an immunosuppressive effect on CD8+ T cells, which decreases the efficacy of CD8+ T cell-targeting immunotherapies. These findings identify βAR signaling as a target for modulation during cancer immunotherapy that may increase therapeutic response and improve patient outcomes. Cancer Immunol Res; 6(1); 98-109. ©2017 AACR.

Abstract 4295: AZ’5576, a selective CDK9 inhibitor, demonstrates in vitro and in vivo activity in diverse preclinical models of non-Hodgkin lymphoma

Abstract Cyclin-dependent kinase 9 (Cdk9) is a serine/threonine kinase that regulates elongation of transcription through phosphorylation of RNA polymerase II at serine 2 (pSer2-RNAPII). Transient inhibition of Cdk9 results in modulation of genes with short-lived transcripts and labile proteins, thereby representing a potential therapeutic opportunity in tumors dependent upon oncogenes fitting these criteria. Mcl1, an anti-apoptotic protein that has been linked to increased survival and chemotherapy resistance in various cancers, and Myc, a proto-oncogenic transcription factor that coordinates diverse transcription programs and is over-expressed, amplified, or translocated in many cancers, are two such oncogenes. AZ’5576 is a potent, highly selective, and orally bioavailable inhibitor of Cdk9 that inhibits Cdk9 enzyme activity with an IC50 &amp;lt;5nM and decreases phosphorylation of Ser2-RNAPII in cells with an IC50 of 96nM. In sensitive cell lines, short-term treatment with AZ’5576 led to a rapid dose- and time-dependent decrease in pSer2-RNAPII with concomitant loss of Mcl1 and Myc mRNA and protein, resulting in the cleavage and activation of caspase 3 and loss of cell viability. Using cell potency and magnitute of caspase activation to define sensitivity, we previously reported that AZ’5576 demonstrates broad in vitro and in vivo activity across hematological malignancies. Initially focusing on acute myeloid leukemia (AML) and multiple myeloma (MM), we have now expanded our scope to encompass non-Hodgkin lymphoma (NHL), which includes diffuse large B-cell lymphoma (DLBCL), Burkitt’s lymphoma (BL), mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia (WM). In vitro screening assays reveal about half (19/35) of NHL cell lines undergo cell death in response to AZ’5576, demonstrating a potency &amp;lt;520nM and a maximum caspase activation &amp;gt;55% after 6 hour treatment. Combining AZ’5576 with a BTK inhibitor further enhances cell death compared to that observed with either single agent alone in BTK inhibitor-sensitive ABC-DLBCL cell lines. Consistent with the in vitro data, significant anti-tumor activity was associated with short-term reduction of pSer2-RNAPII tumor levels after intermittent dosing of AZ’5576 in a mouse xenograft model of ABC-DLBCL cell line OCILY10. Similarly, combining AZ’5576 with a BTK inhibitor in vivo drove tumors into regression (combination=199% TGI) compared to the partial TGI from either single agent alone (AZ’5576=79% TGI, Acalabrutinib=58% TGI) in the OCILY10 xenograft model. Finally, in an agressive Eµ-Myc transgenic mouse model of B-cell lymphoma, AZ’5576 treatment results in potent induction of apoptosis in vitro and a greater than 50 day increase in median overall survival in vivo. Together, these results highlight the therapeutic potential for selective CDK9 inhibition in the treatment of patients with non-Hodgkin lymphoma. Citation Format: Justin Cidado, Theresa Proia, Gareth Gregory, Izabela Todorovski, Scott Boiko, Maryann San Martin, Ricky Johnstone, Lisa Drew. AZ’5576, a selective CDK9 inhibitor, demonstrates in vitro and in vivo activity in diverse preclinical models of non-Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4295. doi:10.1158/1538-7445.AM2017-4295

Opposing impact of AID deficiency on BCL-2 and IL-6 driven B-lymphoma development in BALB/c mice

Activation-induced cytidine deaminase (AID), an essential enzymatic activity required for somatic hypermutation and immunoglobulin class switch recombination in the course of normal B-lymphocyte development, has been implicated in the initiation and promotion of malignant B-cell tumors by virtue of a complex mechanism that includes the generation of oncogene-activating genomic rearrangements and the introduction of point mutations in cancer genes. Here, we use transgenic mouse models of B-cell lymphoma driven by the pro-inflammatory cytokine, interleukin 6 (IL-6), or the survival-enhancing oncoprotein, B-cell leukemia 2 (BCL-2), to evaluate the impact of loss of AID on neoplastic B-cell development. We show that AID deficiency accelerates BCL-2 induced lymphoma but delays IL-6 induced lymphoma. This led us to conclude that AID may function as tumor suppressor or tumor promoter, depending on the genetic context. Elucidating the mechanism of AID's dual function during malignant B-cell transformation may be important for new approaches to tumor treatment and prevention.

How to avoid being eaten: a lymphoma's defense

In this issue of Blood, Lykken et al used an immunocompetent mouse model of B-cell lymphoma to discover an interesting new way in which these malignant cells can avoid being killed in the presence of anti-CD20 antibodies.

BET bromodomain inhibitors synergize with ATR inhibitors to induce DNA damage, apoptosis, senescence-associated secretory pathway and ER stress in Myc-induced lymphoma cells.

Inhibiting the bromodomain and extra-terminal (BET) domain family of epigenetic reader proteins has been shown to have potent anti-tumoral activity, which is commonly attributed to suppression of transcription. In this study, we show that two structurally distinct BET inhibitors (BETi) interfere with replication and cell cycle progression of murine Myc-induced lymphoma cells at sub-lethal concentrations when the transcriptome remains largely unaltered. This inhibition of replication coincides with a DNA-damage response and enhanced sensitivity to inhibitors of the upstream replication stress sensor ATR in vitro and in mouse models of B-cell lymphoma. Mechanistically, ATR and BETi combination therapy cause robust transcriptional changes of genes involved in cell death, senescence-associated secretory pathway, NFkB signaling and ER stress. Our data reveal that BETi can potentiate the cell stress and death caused by ATR inhibitors. This suggests that ATRi can be used in combination therapies of lymphomas without the use of genotoxic drugs.