Mouse Lymphoma Research Articles

Optimisation of Animal Handing and Timing of 2-deoxy-2-[18F]fluoro-D-glucose PET Tumour Imaging in Mice

Abstract Purpose In humans, 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) tumour-to-background contrast continues to increase long after a typical uptake period of 45 – 60 min. Similar studies have not been performed in mice and the static imaging time point for most studies is arbitrarily set at 30 – 60 min post-injection of [18F]FDG. Ideally, static PET imaging should be performed after the initial period of rapid uptake but this period has not been defined in mice, with previous dynamic studies in mice being limited to 60 min. This study aimed to define the kinetics of [18F]FDG biodistribution over periods of 3 – 4 h in different murine tumour models, both subcutaneous and autochthonous, and to further refine fasting and warming protocols used prior to imaging. Procedures Dynamic [18F]FDG PET-CT scans lasting 3 or 4 h were performed with C57BL/6 J and Balb/c nude mice bearing subcutaneous EL4 murine T-cell lymphoma and Colo205 human colorectal tumours, respectively, and with transgenic Eμ-Myc lymphoma mice. Prior to [18F]FDG injection, four combinations of different animal handling conditions were used: warming for 1 h at 31 °C; maintenance at room temperature (20 – 24 °C), fasting for 6 – 10 h and a fed state. Results Tumour mean standardised uptake value (SUVmean) peaked at 147 ± 48 min post injection in subcutaneous tumours and 74 ± 31 min in autochthonous Eμ-Myc lymphomas. The tumour-to-blood ratio (TBR) peaked at 171 ± 57 and 83 ± 33 min in subcutaneous and autochthonous Eμ-Myc tumours, respectively. Fasting increased tumour [18F]FDG uptake and suppressed myocardial uptake in EL4 tumour-bearing mice. There was a good correlation between tumour SUVmean and Ki calculated using an input function (IDIF) derived from the inferior vena cava. Conclusions Delayed static [18F]FDG-PET imaging (> 60 min) in both autochthonous and subcutaneous tumours in improved tumour-to-background contrast and increased reproducibility.

Enhancing anti-EGFRvIII CAR T cell therapy against glioblastoma with a paracrine SIRPγ-derived CD47 blocker

A significant challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is its immunosuppressive microenvironment, which is densely populated by protumoral glioma-associated microglia and macrophages (GAMs). Myeloid immune checkpoint therapy targeting the CD47-signal regulatory protein alpha (SIRPα) axis induces GAM phagocytic function, but CD47 blockade monotherapy is associated with toxicity and low bioavailability in solid tumors. In this work, we engineer a CAR T cell against epidermal growth factor receptor variant III (EGFRvIII), constitutively secreting a signal regulatory protein gamma-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eradicate orthotopic EGFRvIII-mosaic GBM in vivo, promoting GAM-mediated tumor cell phagocytosis. In a subcutaneous CD19+ lymphoma mouse model, anti-CD19-SGRP CAR T cell therapy is superior to conventional anti-CD19 CAR T. Thus, combination of CAR and SGRP eliminates bystander tumor cells in a manner that could overcome main mechanisms of CAR T cell therapy resistance, including immune suppression and antigen escape.

Antibody blockade of the PSGL-1 immune checkpoint enhances T-cell responses to B-cell lymphoma.

Despite advancements in cancer immunotherapy, most lymphomas remain unresponsive to checkpoint inhibitors. P-selectin glycoprotein ligand-1 (PSGL-1), recently identified as a promoter of T-cell exhaustion in murine melanoma models, has emerged as a novel immune checkpoint protein and promising immunotherapeutic target. In this study, we investigated the potential of PSGL-1 antibody targeting in B-cell lymphoma. Using allogeneic co-culture systems, we demonstrated that targeted antibody interventions against human PSGL-1 enhanced T-cell activation and effector cytokine production in response to lymphoma cells. Moreover, in vitro treatment of primary lymphoma cell suspensions with PSGL-1 antibody resulted in increased activation of autologous lymphoma-infiltrating T cells. Using the A20 syngeneic B-cell lymphoma mouse model, we found that PSGL-1 antibody treatment significantly slowed tumor development and reduced the endpoint tumor burden. This antitumoral effect was accompanied by augmented tumor infiltration of CD4+ and CD8+ T cells and reduced infiltration of regulatory T cells. Finally, anti-PSGL-1 administration enhanced the expansion of CAR T cells previously transferred to mice bearing the aggressive Eμ-Myc lymphoma cells and improved disease control. These results demonstrate that PSGL-1 antibody blockade bolsters T-cell activity against B-cell lymphoma, suggesting a potential novel immunotherapeutic approach for treating these malignancies.

Modeling NK-cell lymphoma in mice reveals its cell-of-origin and microenvironmental changes and identifies therapeutic targets

Extranodal NK/T-cell lymphoma (ENKTCL) is an Epstein-Barr virus (EBV)-related neoplasm preferentially involving the upper aerodigestive tract. Here we show that NK-cell-specific Trp53 disruption in mice leads to the development of NK-cell lymphomas after long latency, which involve not only the hematopoietic system but also the salivary glands. Before tumor onset, Trp53 knockout causes extensive gene expression changes, resulting in immature NK-cell expansion, exclusively in the salivary glands. Both human and murine NK-cell lymphomas express tissue-resident markers, suggesting tissue-resident NK cells as their cell-of-origin. Murine NK-cell lymphomas show recurrent Myc amplifications and upregulation of MYC target gene signatures. EBV-encoded latent membrane protein 1 expression accelerates NK-cell lymphomagenesis and causes diverse microenvironmental changes, particularly myeloid propagation, through interferon-γ signaling. In turn, myeloid cells support tumor cells via CXCL16-CXCR6 signaling and its inhibition is effective against NK-cell tumors in vivo. Remarkably, KLRG1-expressing cells expand in the tumor and are capable of repopulating tumors in secondary recipients. Furthermore, targeting KLRG1 alone or combined with MYC inhibition using an eIF4 inhibitor is effective against NK-cell tumors. Therefore, our observations provide insights into the pathogenesis and highlight potential therapeutic targets, including CXCL16, KLRG1, and MYC, in ENKTCL, which can help improve its diagnostic and therapeutic strategies.

PD-L1 blockade immunotherapy rewires cancer-induced emergency myelopoiesis.

Immune checkpoint blockade (ICB) immunotherapy has revolutionized cancer treatment, demonstrating exceptional clinical responses in a wide range of cancers. Despite the success, a significant proportion of patients still fail to respond, highlighting the existence of unappreciated mechanisms of immunotherapy resistance. Delineating such mechanisms is paramount to minimize immunotherapy failures and optimize the clinical benefit. In this study, we treated tumour-bearing mice with PD-L1 blockage antibody (aPD-L1) immunotherapy, to investigate its effects on cancer-induced emergency myelopoiesis, focusing on bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). We examined the impact of aPD-L1 treatment on HSPC quiescence, proliferation, transcriptomic profile, and functionality. Herein, we reveal that aPD-L1 in tumour-bearing mice targets the HSPCs in the BM, mediating their exit from quiescence and promoting their proliferation. Notably, disruption of the PDL1/PD1 axis induces transcriptomic reprogramming in HSPCs, observed in both individuals with Hodgkin lymphoma (HL) and tumour-bearing mice, shifting towards an inflammatory state. Furthermore, HSPCs from aPDL1-treated mice demonstrated resistance to cancer-induced emergency myelopoiesis, evidenced by a lower generation of MDSCs compared to control-treated mice. Our findings shed light on unrecognized mechanisms of action of ICB immunotherapy in cancer, which involves targeting of BM-driven HSPCs and reprogramming of cancer-induced emergency myelopoiesis.

A Multifunctional Biomimetic Nanoplatform for Dual Tumor Targeting-Assisted Multimodal Therapy of Colon Cancer.

The biomimetic nanoparticles (NPs) possessing abilities of tumor targeting and multimodal therapy show great potential for efficient combat of colon cancer. Herein, we developed a multifunctional biomimetic nanoplatform (Fe3O4@PDA@CaCO3-ICG@CM) based on CaCO3-modified magnetic polydopamine (PDA) loaded with indocyanine green (ICG), which was encapsulated by a mouse lymphoma cell (EL4) membrane (CM) expressing functional proteins (i.e., lymphocyte function-associated antigen 1, LFA-1; transforming growth factor-β receptor, TGF-βR; programmed cell death protein 1, PD-1; and factor related apoptosis ligand, FasL). Under magnetic attraction and LFA-1/PD-1-mediated endocytosis, Fe3O4@PDA@CaCO3-ICG@CM efficiently targeted CT26 colon tumor cells. The released calcium ion (Ca2+) from the NPs triggered by acidic tumor microenvironment, the enhanced photothermal effect contributed by the combination of PDA and ICG, and FasL's direct killing effect together induced tumor cells apoptosis. Moreover, the apoptosis of CT26 cells induced immunogenic cell death (ICD) to promote the maturation of dendritic cells (DCs) to activate CD4+/CD8+ T cells, thereby fighting against tumor cells, which could further be boosted by programmed death-ligand 1 (PD-L1) blockage and transforming growth factor-β (TGF-β) scavenging by Fe3O4@PDA@CaCO3-ICG@CM. As a result, in vivo satisfactory therapeutic effect was observed for CT26 tumor bearing-mice treated with Fe3O4@PDA@CaCO3-ICG@CM under laser irradiation and magnetic attraction, which could eradicate primary tumors and restrain distant tumors through dual tumor targeting-assisted multimodal therapy and eliciting adaptive antitumor immune response, generating the immune memory for inhibiting tumor metastasis and recurrence. Taken together, the multifunctional biomimetic nanoplatform exhibits superior antitumor effects, providing an insightful strategy for the field of nanomaterial-based treatment of cancer.

Cancer cell-intrinsic biosynthesis of itaconate promotes tumor immunogenicity.

The Krebs cycle byproduct itaconate has recently emerged as an important metabolite regulating macrophage immune functions, but its role in tumor cells remains unknown. Here, we show that increased tumor-intrinsic cis-aconitate decarboxylase (ACOD1 or CAD, encoded by immune-responsive gene 1, Irg1) expression and itaconate production promote tumor immunogenicity and anti-tumor immune responses. Furthermore, we identify thimerosal, a vaccine preservative, as a specific inducer of IRG1 expression in tumor cells but not in macrophages, thereby enhancing tumor immunogenicity. Mechanistically, thimerosal induces itaconate production through a ROS-RIPK3-IRF1 signaling axis in tumor cells. Further, increased IRG1/itaconate upregulates antigen presentation-related gene expression via promoting TFEB nuclear translocation. Intratumoral injection of thimerosal induced itaconate production, activated the tumor immune microenvironment, and inhibited tumor growth in a T cell-dependent manner. Importantly, IRG1 deficiency markedly impaired tumor response to thimerosal treatment. Furthermore, itaconate induction by thimerosal potentiates the anti-tumor efficacy of adoptive T-cell therapy and anti-PD1 therapy in a mouse lymphoma model. Hence, our findings identify a new role for tumor intrinsic IRG1/itaconate in promoting tumor immunogenicity and provide a translational means to increase immunotherapy efficacy.

Increased CCL2/CCR2 axis promotes tumor progression by increasing M2 macrophages in MYC/BCL2 double-expressor DLBCL

AbstractThe pathogenesis of myelocytomatosis oncogene (MYC) and B-cell lymphoma 2 (BCL2) double-expressor diffuse large B-cell lymphoma (DE-DLBCL) remains unclear. To investigate how MYC and BCL2 contribute to tumor aggressiveness, we analyzed tumors from 14 patients each with DE-DLBCL and non–DE-DLBCL using whole transcriptome sequencing. Validation was performed using publicly available data sets, tumor tissues from 126 patients, DLBCL cell lines, and a syngeneic mouse lymphoma model. Our transcriptome analysis revealed significantly elevated messenger RNA levels of C-C motif chemokine ligand 2 (CCL2) and C-C chemokine receptor type 2 (CCR2) in DE-DLBCLs when compared with non–DE-DLBCLs (adjusted P value < .05). Transcriptomic analysis of public data sets and immunohistochemistry corroborated these findings, indicating increased levels of M2 macrophages but a reduction in T-cell infiltration in DE-DLBCLs when compared with non–DE-DLBCLs (all P < .05). CCR2 expression was observed mainly in tumor-infiltrating macrophages and not in DLBCL cells. Increased expression of CCL2 and CCR2 was significantly associated with a poor prognosis in patients with DLBCL. In the in vitro analyses, MYChigh/BCL2high DLBCL cells showed higher CCL2 expression and secretion than MYClow/BCL2low cells. MYC and BCL2 increased CCL2 expression and secretion by upregulation of nuclear factor κB p65 in DLBCL cells, and CCL2 promoted M2 polarization of macrophages. In a mouse lymphoma model, CCL2 contributed to the immunosuppressive microenvironment and tumor growth of MYChigh/BCL2high tumors. We demonstrated that the increased CCL2/CCR2 axis confers aggressiveness to DE-DLBCL by increasing M2 polarization and can be a potential therapeutic target.

Tumor-induced natural killer cell dysfunction is a rapid and reversible process uncoupled from the expression of immune checkpoints.

Natural killer (NK) cells often become dysfunctional during tumor progression, but the molecular mechanisms underlying this phenotype remain unclear. To explore this phenomenon, we set up mouse lymphoma models activating or not activating NK cells. Both tumor types elicited type I interferon production, leading to the expression of a T cell exhaustion-like signature in NK cells, which included immune checkpoint proteins (ICPs). However, NK cell dysfunction occurred exclusively in the tumor model that triggered NK cell activation. Moreover, ICP-positive NK cells demonstrated heightened reactivity compared to negative ones. Furthermore, the onset of NK cell dysfunction was swift and temporally dissociated from ICPs induction, which occurred as a later event during tumor growth. Last, NK cell responsiveness was restored when stimulation was discontinued, and interleukin-15 had a positive impact on this reversion. Therefore, our data demonstrate that the reactivity of NK cells is dynamically controlled and that NK cell dysfunction is a reversible process uncoupled from the expression of ICPs.

Glutathione overproduction mediates lymphoma initiating cells survival and has a sex-dependent effect on lymphomagenesis

Lymphoid tumor patients often exhibit resistance to standard therapies or experience relapse post-remission. Relapse is driven by Tumor Initiating Cells (TICs), a subset of tumor cells capable of regrowing the tumor and highly resistant to therapy. Growing cells in 3D gels is a method to discern tumorigenic cells because it strongly correlates with tumorigenicity. The finding that TICs, rather than differentiated tumor cells, grow in 3D gels offers a unique opportunity to unveil TIC-specific signaling pathways and therapeutic targets common to various cancer types. Here, we show that culturing lymphoid cells in 3D gels triggers reactive oxygen species (ROS) production, leading to non-tumor lymphoid cell death while enabling the survival and proliferation of a subset of lymphoma/leukemia cells, TICs or TIC-like cells. Treatment with the antioxidant N-acetylcysteine inhibits this lethality and promotes the growth of primary non-tumor lymphoid cells in 3D gels. A subset of lymphoma cells, characterized by an increased abundance of the antioxidant glutathione, escape ROS-induced lethality, a response not seen in non-tumor cells. Reducing glutathione production in lymphoma cells, either through pharmacological inhibition of glutamate cysteine ligase (GCL), the enzyme catalyzing the rate-limiting step in glutathione biosynthesis, or via knockdown of GCLC, the GCL catalytic subunit, sharply decreased cell growth in 3D gels and xenografts. Tumor cells from B-cell lymphoma/leukemia patients and λ-MYC mice, a B-cell lymphoma mouse model, overproduce glutathione. Importantly, pharmacological GCL inhibition hindered lymphoma growth in female λ-MYC mice, suggesting that this treatment holds promise as a therapeutic strategy for female lymphoma/leukemia patients.

Evaluation of mRNA lipoplexes prepared using modified ethanol injection method as a tumor vaccine

We have previously demonstrated that messenger RNA (mRNA) lipoplexes composed of N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and polyethylene glycol-cholesteryl ether (PEG-Chol) exhibited high protein expression in the lungs and spleen of mice after intravenous injection and induced high levels of antigen-specific IgG1 upon immunization. In this study, we optimized PEG modification in mRNA lipoplexes to reduce mRNA accumulation in the lungs and evaluated the suppression of tumor growth in mice bearing mouse lymphoma E.G7-ovalbumin (OVA) tumors by immunizing them with an intravenous injection of OVA mRNA lipoplexes. PEGylation of mRNA lipoplexes with 3 mol% PEG-Chol (LP-DC-1-16-3PCL) prevented agglutination of erythrocytes and reduced accumulation in the lungs. Intravenous injection of LP-DC-1-16-3PCL lipoplexes containing OVA mRNA into mice induced high levels of anti-OVA IgG1 (83,000 mU/mL) in serum, and exhibited a high cytotoxic activity (97%) against E.G7-OVA cells by the splenocytes of mice. Furthermore, immunization with LP-DC-1-16-3PCL lipoplexes containing OVA mRNA suppressed E.G7-OVA tumor growth compared to control mRNA. Based on these results, LP-DC-1-16-3PCL lipoplexes may be an effective mRNA vaccine for inducing antibody- and cytotoxic cell-mediated immune responses to tumors through intravenous injection.

Tethered IL15-IL15Rα augments antitumor activity of CD19 CAR-T cells but displays long-term toxicity in an immunocompetent lymphoma mouse model

BackgroundAdoptive cell therapy using genetically modified T cells to express chimeric antigen receptors (CAR-T) has shown encouraging results, particularly in certain blood cancers. Nevertheless, over 40% of B cell malignancy...

Novel OX40 and 4-1BB derived spacers enhance CD30 CAR activity and safety in CD30 positive lymphoma models

The chimeric antigen receptor (CAR) derived from the CD30 specific murine antibody, HRS-3, has produced promising clinical efficacy with a favorable safety profile in the treatment of relapsed or refractory CD30-positive lymphomas. However, persistence of the autologous CAR-T cells was brief, and many patients relapsed a year after treatment. The lack of persistence may be attributed to the use of a wild-type immunoglobulin (Ig)G1 spacer that can associate with Fc receptors. We first identified the cysteine-rich domain (CRD) 5 of CD30 as the primary binding epitope of HRS-3 and armed with this insight, attempted to improve the HRS-3 CAR functionality with a panel of novel spacer designs. We demonstrate that HRS-3 CARs with OX40 and 4-1BB derived spacers exhibited similar anti-tumor efficacy, circumvented interactions with Fc receptors, and secreted lower levels of cytokines invitro than a CAR employing the IgG1 spacer. Humanization of the HRS-3 scFv coupled with the 4-1BB spacer preserved potent on-target, on-tumor efficacy, and on-target, off-tumor safety. In a lymphoma mouse model of high tumor burden, Tcells expressing humanized HRS-3 CD30.CARs with the 4-1BB spacer potently killed tumors with low levels of circulating inflammatory cytokines, providing a promising candidate for future clinical development in the treatment of CD30-positive malignancies.

Expression of VISTA regulated via IFN-γ governs endogenous T-cell function and exhibits correlation with the efficacy of CD19 CAR-T cell treated B-malignant mice

BackgroundDespite continuous improvements in the new target and construction of chimeric antigen receptor (CAR)-T, relapse remains a significant challenge following CAR-T therapy. Tumor microenvironment (TME) strongly correlates with the efficacy...

Effect of AVM0703 treatment on the limitations of R-CHOP and survival in a model applicable to ABC subtype DLBCL.

e19060 Background: The current treatment regimen for DLBCL consisting of rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone (RCHOP), is challenged by molecular and prognostic characterization of DLBCL. Activated B-cell (ABC) lymphoma has a significantly worse prognosis (5 yr OS rate: 45% - 56%) than germinal center B-cell (GCB) lymphoma (78% - 80%) after RCHOP treatment. Around 40% of patients ultimately relapse and/or develop primary refractory disease, cardiotoxicity and long-term secondary malignancies. Efforts are being made to improve the RCHOP backbone by including dose intensification and by the addition of new agents to increase the efficacy and reduced toxicity of RCHOP (McKelvey EM Cancer 1976; Wilson WH Blood 2002; Molina TJ JCO 2014; Coiffier B Hematology 2016). AVM0703, an immunomodulatory drug, mobilizes bispecific γδ and inv-TCR double-positive novel T and NKT cells. In a previous study employing an aggressive, immune resistant A20 mouse lymphoma model (Bascuas T J Transl Med. 2016), neoadjuvant AVM0703 was additive/synergistic, when cyclophosphamide/fludarabine (CyFlu) was dosed 3 and 24 hr later, with no additional toxicities. In the present study we combined AVM0703 with RCHOP to evaluate the efficacy in A20 mice model. Methods: 10 week old Balb/c mice were injected with 2x106 murine A20 B-cells in the flank and were randomized (1:1 male: female) into 1) Placebo 2) 3 cycles RCHOP 3) 3 cycles AVM0703 (18mg/kg HED) 4) 3 cycles AVM0703+RCHOP, when tumor size reached ~100 mm3. Mice who achieved complete remission (CR) were re-challenged at 21-25 wks later. Pre and post re-challenge, presence of memory cells was analyzed using flow cytometer. All mice were monitored continuously as per the IACUC protocol. Results: Mice treated with the combination of AVM0703+RCHOP achieved superior CR compared to RCHOP alone, which was not curative in any mice. In first phase of the study, 3 out of 11 AVM0703 +RCHOP treated mice showed CR after only 1-cycle. In second phase, 2 of 12 mice survived in CR for &gt;150 days after 3 cycles, whereas no RCHOP mice survived beyond 2 cycles. AVM0703+RCHOP treated mice in long-term CR, had memory cells (CD44hi-Cd62low) pre- and post re-challenge, and no A20 engraftment, suggesting strong immunological memory induced by AVM0703+RCHOP. Conclusions: AVM+RCHOP demonstrated anti-tumor activity in A20 lymphoma mice, with reduced toxicity, compared to RCHOP. Larger preclinical studies are underway to confirm the activity of AVM0703+RCHOP against RCHOP resistant ABC DLBCL, and the potential to reduce total RCHOP exposure to only 3 cycles while maintaining or improving efficacy. [Table: see text]

Synergistic effects of RPT1G, a first-in-class small molecule NAMPT inhibitor, with venetoclax and olaparib in hematological malignancies.

e18512 Background: MultipleBCL-2 and PARP inhibitors are currently in clinical trials, or have been approved for use, in hematological malignancies and solid tumors. Venetoclax, an inhibitor of the apoptosis regulator B-cell lymphoma-2 (BCL-2), has activity in patients with chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). PARP inhibitors, such as Olaparib, exploit synthetic lethality in homologous recombination-deficient tumors, notably in breast and ovarian cancers. However, patients treated with BCL-2 or PARP inhibitors often face relapse and refractory disease, requiring alternative strategies to counter adaptive responses. Nicotinamide Phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway, is upregulated in malignancies and plays a key role in cancer metabolism (e.g., Warburg effect). NAMPT makes nicotinamide mononucleotide which is then converted to NAD+, while PARP enzymes consume NAD+, placing NAMPT directly upstream of PARP. NAMPT overexpression is associated with Venetoclax resistance in leukemia patients, highlighting the link between NAD+, mitochondrial metabolism, and BCL-2 inhibition. Consequently, NAMPT inhibition may synergize with BCL-2 or PARP inhibitors, potentially enhancing 1st-line of treatment outcomes and re-sensitizing refractory cancer cells to BCL-2 or PARP inhibition. Methods: In this study, we investigated the combination effects of Venetoclax and Olaparib with RPT1G, a novel hyperbolic NAMPT inhibitor that simultaneously inhibits and stabilizes NAMPT in a catalytically active state. RPT1G disrupts NAMPT activity without ever turning it off completely avoiding the severe on-target toxicities seen with complete NAMPT inhibitors. Several cell lines, including OCI-AML3 and MV4;11 (AML), RS4;11 (ALL), and RL (diffuse large B-cell lymphoma) were treated with RPT1G and either Venetoclax or Olaparib to determine the degree of drug interactions. In a follow up in vivo study, we treated mice in a human xenograft model using semi-resistant MV4;11 cells with RPT1G alone or in combination with Venetoclax to measure the ability of RPT1G to enhance the effects of Venetoclax. Results: RPT1G achieves significant single-agent efficacy in leukemia and lymphoma mouse xenograft models. When RTP1G is used in combination with either Venetoclax or Olaparib significant synergy is seen across a broad range of concentrations in vitro. Consistent with in vitro synergy results, in a xenograft model of human AML, mice treated with a combination of RPT1G and Venetoclax show enhanced efficacy as compared to Venetoclax alone. Conclusions: Combination of RPT1G with either Venetoclax or Olaparib synergistically enhances killing of leukemic cells and presents as an effective strategy to improve treatment outcomes for patients with diverse malignancies.

Caspase-resistant ROCK1 expression prolongs survival of Eµ-Myc B cell lymphoma mice.

Apoptosis is characterized by membrane blebbing and apoptotic body formation. Caspase cleavage of ROCK1 generates an active fragment that promotes actin-myosin-mediated contraction and membrane blebbing during apoptosis. Expression of caspase-resistant non-cleavable ROCK1 (Rock1 NC) prolonged survival of mice that rapidly develop B cell lymphomas due to Eµ-Myc transgene expression. Eµ-Myc; Rock1 NC mice had significantly fewer bone marrow cells relative to those in Eµ-Myc mice expressing wild-type ROCK1 (Rock1 WT), which was associated with altered cell cycle profiles. Circulating macrophage numbers were lower in Eµ-Myc; Rock1 NC mice, but there were higher levels of bone marrow macrophages, consistent with spontaneous cell death in Eµ-Myc; Rock1 NC mouse bone marrows being more inflammatory. Rock1 WT recipient mice transplanted with pre-neoplastic Eµ-Myc; Rock1 NC bone marrow cells survived longer than mice transplanted with Eµ-Myc; Rock1 WT cells, indicating that the survival benefit was intrinsic to the Eµ-Myc; Rock1 NC bone marrow cells. The results suggest that the apoptotic death of Eµ-Myc; Rock1 NC cells generates a proliferation-suppressive microenvironment in bone marrows that reduces cell numbers and prolongs B cell lymphoma mouse survival.

TYM-3-98, a novel selective inhibitor of PI3Kδ, demonstrates promising preclinical antitumor activity in B-cell lymphomas

AimsPI3Kδ is expressed predominately in leukocytes and is commonly found to be aberrantly activated in human B-cell lymphomas. Although PI3Kδ has been intensively targeted for discovering anti-lymphoma drugs, the application of currently approved PI3Kδ inhibitors has been limited due to unwanted systemic toxicities, thus warranting the development of novel PI3Kδ inhibitors with new scaffolds. Main methodsWe designed TYM-3-98, an indazole derivative, and evaluated its selectivity for all four PI3K isoforms, as well as its efficacy against various B-cell lymphomas both in vitro and in vivo. Key findingsWe identified TYM-3-98 as a highly selective PI3Kδ inhibitor over other PI3K isoforms at both molecular and cellular levels. It showed superior antiproliferative activity in several B-lymphoma cell lines compared with the approved first-generation PI3Kδ inhibitor idelalisib. TYM-3-98 demonstrated a concentration-dependent PI3K/AKT/mTOR signaling blockage followed by apoptosis induction. In vivo, TYM-3-98 showed good pharmaceutical properties and remarkably reduced tumor growth in a human lymphoma xenograft model and a mouse lymphoma model. SignificanceOur findings establish TYM-3-98 as a promising PI3Kδ inhibitor for the treatment of B-cell lymphoma.

Latent Epstein-Barr virus infection collaborates with Myc over-expression in normal human B cells to induce Burkitt-like Lymphomas in mice.

Epstein-Barr virus (EBV) is an important cause of human lymphomas, including Burkitt lymphoma (BL). EBV+ BLs are driven by Myc translocation and have stringent forms of viral latency that do not express either of the two major EBV oncoproteins, EBNA2 (which mimics Notch signaling) and LMP1 (which activates NF-κB signaling). Suppression of Myc-induced apoptosis, often through mutation of the TP53 (p53) gene or inhibition of pro-apoptotic BCL2L11 (BIM) gene expression, is required for development of Myc-driven BLs. EBV+ BLs contain fewer cellular mutations in apoptotic pathways compared to EBV-negative BLs, suggesting that latent EBV infection inhibits Myc-induced apoptosis. Here we use an EBNA2-deleted EBV virus (ΔEBNA2 EBV) to create the first in vivo model for EBV+ BL-like lymphomas derived from primary human B cells. We show that cord blood B cells infected with both ΔEBNA2 EBV and a Myc-expressing vector proliferate indefinitely on a CD40L/IL21 expressing feeder layer in vitro and cause rapid onset EBV+ BL-like tumors in NSG mice. These LMP1/EBNA2-negative Myc-driven lymphomas have wild type p53 and very low BIM, and express numerous germinal center B cell proteins (including TCF3, BACH2, Myb, CD10, CCDN3, and GCSAM) in the absence of BCL6 expression. Myc-induced activation of Myb mediates expression of many of these BL-associated proteins. We demonstrate that Myc blocks LMP1 expression both by inhibiting expression of cellular factors (STAT3 and Src) that activate LMP1 transcription and by increasing expression of proteins (DNMT3B and UHRF1) known to enhance DNA methylation of the LMP1 promoters in human BLs. These results show that latent EBV infection collaborates with Myc over-expression to induce BL-like human B-cell lymphomas in mice. As NF-κB signaling retards the growth of EBV-negative BLs, Myc-mediated repression of LMP1 may be essential for latent EBV infection and Myc translocation to collaboratively induce human BLs.

Abstract LB081: Novel targeted oligonucleotide-based telomere disruptor for immunotherapy of diffuse large B-cell lymphoma

Abstract Telomerase (TERT) is an enzyme critical for carcinogenesis expressed in about 90% of human cancers, including aggressive diffuse large B-cell lymphoma (DLBCL). Targeting TERT proven difficult due to on-target toxicities to non-malignant hematopoietic stem cells and potentially to activated immune effector cells. To overcome these challenges, we developed novel TLR9+ cell-selective CpG-oligodeoxynucleotides (ODNs) to deliver telomere-damaging nucleoside analogue, 6-thio-2’-deoxy-guanosine (6tdG), into target B cell lymphoma cells in vitro and in vivo. Human and mouse TLR9+/TERT+ lymphoma cells rapidly internalized CpG(6tdG)-oligonucleotides through active endocytosis. 6tdG molecules were released from the 3’ end of the oligonucleotide into cytosol and cell nucleus. TERT-mediated 6tdG incorporation into telomeres, led to DNA damage and lymphoma cell death. In vitro, CpG(6tdG)-oligonucleotides showed potency in targeting variety of human DLBCL cells in nanomolar range without detectable toxicity to non-malignant TERT— cells or human TERT+ hematopoietic stem cells and activated T cells. Importantly, the repeated systemic administration of CpG(6tdG)-oligonucleotides was well-tolerated by humanized hCD34/NOG mice, without detectable decrease in the percentage of hematopoietic stem cells, myeloid cells or T cells although the numbers of B cells were partly reduced as expected. Intravenously injected CpG(6tdG)-oligonucleotides, but not 6tdG nucleoside, had potent and dose-dependent antitumor effects against xenotransplanted models of human GC- or ABC-subtypes of DLBCL, such as OCI-Ly18 or OCI-Ly3, respectively. Furthermore, we assessed CpG(6tdG)-oligonucleotide activity against aggressive, syngeneic TERT+ Eμ-myc/15A lymphoma in immunocompetent mice. CpG(6tdG)-oligonucleotide, but not the CpG-oligonucleotide alone, 6tdG alone or co-injection of thereof, was effective in triggering regression of established 15A tumors with long-term survival of the majority of mice. The effect of CpG(6tdG)-oligonucleotide correlated with the activation of tumor antigen-specific T cell-mediated antitumor immune responses. In contrast, when tested in immunodeficient mice, CpG(6tdG)-oligonucleotide induced significant but transient regression of 15A lymphoma. These results suggest that the oligonucleotide elicits two-pronged activity triggering direct lymphoma cell cytotoxicity and also immune-mediated antitumor effects. Overall, this cell-selective strategy could provide an effective and safer strategy against aggressive TERT+ DLBCL cells with potential to engage and support T cell-based antitumor immunity. Citation Format: Chunsong Yu, Yong Liang, Piotr Swiderski, Larry Kwak, Marcin Kortylewski. Novel targeted oligonucleotide-based telomere disruptor for immunotherapy of diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB081.