Epigenetics, especially DNA methylation, is a key molecular regulator of gene expression. This regulation is important for cytotoxic cell activation (i.e., T-cells and NK cells) and is crucial for ...the clearance of malignant cells. Immune checkpoint (IC) events mediate cytotoxicity, one IC receptor involved in the process is CD155. Such receptor interacts primarily with TIGIT on cytotoxic cells and induces the inhibition of cytotoxicity. Multiple myeloma (MM) represents the 10% of all hematological malignancies and it has been described that MM cells hinder cytotoxic cell activation by inhibition and exhaustion. Furthermore, novel immunotherapeutic strategies such as Bispecific T-cell Engagers (BiTE) and Chimeric Antigen Receptor (CAR) therapies are being evaluated in MM treatment, with promising results. Nevertheless, they will confront a hostile environment. This is a very active field with frequent descriptions of new targets and therapeutic approaches being studied each day. We investigated whether CD155 is epigenetically regulated in MM and its importance in inducing inhibition of anti-tumor response in the context of novel immunotherapies through the CD155/TIGIT axis. To achieve our objectives, we focused on the promoter region of CD155 (the region up to 1500 base pairs upstream of the TSS) and characterized it at DNA, RNA and protein levels through bisulfite sequencing, quantitative PCR, and flow cytometry. We confirmed CD155 as an epigenetically regulated gene, thus demonstrating an existing correlation between CD155 promoter methylation and gene expression. In vitro treatment with azacytidine was performed to assess the active repression of CD155 expression by DNA methylation. Six different MM cell lines were characterized (two not expressing: AMO-1, KMS-12-BM, four expressing: RPMI-8226, MM.1S, EJM and JJN-3). From there, we created CD155 depletion models on expressing cell lines by short hairpin RNA interference and developed an in vitro coculture system with T-cells (from healthy donors). We observed a loss of expression between the 60% to 90% (when compared with controls). Depletion of CD155 did not significantly impact cell growth, apoptosis, or cell cycle. On the coculture with healthy T-cells, more cytotoxicity was detected against CD155-depleted cells (in RPMI-8226 Mann-Whitney P=0.020; JJN-3 and EJM P<0.001). To incorporate novel immunotherapeutic strategies, we carried out the same cocultures with 100pg/mL of anti-BCMA/CD3 BiTE, against anti-BCMA CAR-T or anti-BCMA CAR-NK cells (for RPMI-8226 and JJN-3 Mann-Whitney P=0.008). Coculture combinations were also performed in the presence of 10μg/ml of neutralizing αTIGIT (RPMI-8226, Mann-Whitney P =0.005; JJN-3 P=0.004) and/or αPD1 antibody. When novel immunotherapy approaches were included in the system, we observed how the depletion of CD155 was related to a cytotoxicity increase in all cases. In the presence of neutralizing αTIGIT, cytotoxicity was increased against CD155-expressing cells, while we observed no difference against CD155-depleted cells. In the presence of αPD1, we saw a significant restoration of T-cell cytotoxicity against both models and the antibody combination showed a synergic effect in CD155 expressing cells. Finally, we analyzed the existence of a correlation between CD155 gene expression and survival in newly diagnosed MM samples from CoMMpass project public data (N= 793). These data supported our results as the newly diagnosed MM patients with higher expression of CD155 had shorter overall survival than lower expressing ones (Logrank test P<0.001). In summary, in the context of MM, the expression of CD155 is regulated by the methylation status of its promoter region. Expression of CD155 effectively reduces cytotoxic cell response, which is mediated by the interaction of CD155 and TIGIT. The addition of αTIGIT and αPD1 validated that the CD155 cytotoxic cell inhibition is mediated in part by TIGIT interaction, independently of PD1/PD-L1 axis. This interaction seems to impact patients treated with current therapies, since their outcome correlates with CD155 expression. Based on our results, novel immunotherapies focused on enhancing immune cytotoxicity will benefit from CD155/TIGIT axis blockade. These results warrant further investigation on CD155 as a biomarker and target for novel immunotherapies for MM and other malignancies.
Introduction CART-cell therapy (CART) has produced a paradigm shift in the treatment of relapsing non-Hodgkin B-cell lymphoma (NHBcL). However, patients continue to relapse. Thus, developing ...strategies to optimize disease surveillance after this therapy is becoming increasingly important to attain disease-free survival and predict a potential relapse in advance. To facilitate its applicability, such strategies should also be minimally invasive. Hence, this study aims to explore the value of CloneSight, a circulating tumor DNA (ctDNA) monitoring approach based on a signature of personalized trackable mutations for the follow-up of NHBcL under CART. Methods A total of 30 NHBcL patients treated with CD19 CART-cell therapy were included in this work. Ten patients were diagnosed with Follicular Lymphoma (FL), and 20 with Diffuse Large B-Cell-Lymphoma (DLBCL). Genomic profiling was performed at relapse using FFPE biopsies and cfDNA liquid biopsies to detect somatic mutations by a custom capture enrichment panel (Twist, USA) covering 134 genes. Sequencing was conducted with the Illumina NextSeq platform. Then, the patient-specific somatic mutations were used as biomarkers for quantifying Minimal Residual Disease (MRD) on liquid biopsy follow-up samples with a sensitivity of 10 −4 (Jiménez-Ubieto A et al., Leukemia 2023). The amplicon-based, patient-specific MRD mini-panel also included primers to quantify the CART Phi construct relative to the albumin control gene. A total of 118 peripheral blood samples were collected. Plasma was isolated at day +7, +14, +30, +60, +90, and before progression (59 DLBCL and 59 FL samples). PET/CT examinations were performed on days +90, +180, +365 and every six months in FL, and the same for DLBCL but additionally on day +30. Results We found 185 trackable somatic mutations suitable for MRD monitoring (mean: 6.2 per patient). The most frequently mutated genes were CREBBP (80%), KMT2D (50%) and EP300 (30%) in FL, and KMT2D (45%), CREBBP (40%), and TP53 (35%) in DLBCL. The applicability of the test (detection of at least one somatic mutation) was 100%. Nineteen patients presented a cfDNA evaluation between days +30 and +60 ( Figure, left). Twelve progressed and ten of them were CloneSight-positive. Patient FL107, negative at day 28, presented a positive result at progression (day 180). For patient DLBCL16, the last sample available was at day +30, but the progression occurred at day 80, highlighting the need for sequential response assessment. Regarding the remaining nine cases in CR, five were negative and two positives by the test (SE 83%, SP 71%, PPV 83%, NPV 67%). Of note, FL101 becomes CloneSight-negative in the next Time-point (TP) available (day 112) and maintains MRD negativity in three more TPs (last at day 907). The remaining discordant case, DLBCL21, has a follow-up of only 60 days. Of note, patient DLBCL3 did not respond to CART but achieved CR and Clonesight-negativity under immune checkpoint inhibitor therapy. The ctDNA surveillance highly correlated with the PET/CT results ( Figure, right). Remarkably, 27 out of 32 PET/CT-negative TPs (84%) were consistent with a CloneSight-negative determination on belong two patients that did not progress. Four of five TPs with PET/CT-negative and CloneSight-positive status were classified as false negative PET/CTs, as those patients finally progressed. On the other hand, 13 out of 16 positive PET/CT samples (81%) were CloneSight-positive and the patient progressed. Of the remaining three PET/CT positive CloneSight-negative, two belong to patients that also progressed. Conclusions: In CAR-T therapy, monitoring with MRD by ctDNA is consolidated as a useful and necessary tool. Our CloneSight test can predict DLBCL and FL outcomes in advance and could be of high utility to detect false positive or negative PET/CT assessments during follow-ups. However, dynamic response assessment is crucial to draw meaningful conclusions. Further research and a larger cohort will be presented at the meeting.
Introduction
Immunotherapy represents a new weapon in the fight against multiple myeloma. Current clinical outcomes using CAR-T cell therapy against multiple myeloma show promise in the eradication ...of the disease. However, these CARs observe relapse as a common phenomenon after treatment due to the reemergence of neoantigens or negative cells. CARs can also be targeted using non-antibody approaches, including the use of receptors, as NKG2D with a wider range of ligands, and ligands to provide target specificity.
Different cell types have been used to improve CAR cell therapy. CAR-T cells are the most commonly used. However, despite its effectiveness, there are still problems to face. The toxicity of the cytokine release syndrome is well known, that is why memory CD45RA- T cells are used to avoid collateral effects, although having lower efficacy. However, CAR-NK cells may have less toxicity and provide a method to redirect these cells specifically to refractory cancer.
The objective of this work was to compare the anti-tumor activity of CAR-T, NKAEs and CAR-NK cells from multiple myeloma patients.
Methods
The activated and expanded NK cells (NKAE) were generated by coculture of peripheral blood mononuclear cells with the previously irradiated CSTX002 cell line. The CD45RA- T cells were obtained by depletion with CD45RA magnetic beads and subsequent culture. The NKAE and T were transduced with an NKG2D-CAR with signaling domains of 4-1BB and CD3z. The expansion of NKAE and the expression of NKG2D-CAR were evaluated by flow cytometry based on the percentage of NK cell population and transduction efficiency by the expression of NKG2D. Europium-TDA release assays (2-4 hours) were performed to evaluate in vitro cytotoxic activity. The antitumor activity of the NKAE (n=4) and CD45RA- (n=4) cells against MM U-266 cells was studied. Methylcellulose cultures were performed to assess the activity against the clonogenic tumor cell. In vivo studies were carried out in NSG mice receiving 5.106 of U266-luc MM cells i.v. injected at day 1. At day 4, mice received 15.106 i.v. injected of either CAR-NKAE or untransduced NKAE cells.
Results
In vitro. The killing activity of primary NKAE cells (n=4) was 86.6% (± 13.9%), considerably higher than that of CD45RA- lymphocytes (16.7% ± 13.6%) from the same patient (n=4). Even CD45RA- T cells from healthy donors (n=4) exhibit lower anti tumoral capacity (28.2% ± 9.7%) than NKAE cells. The transduction with an NKG2D CAR (MOI=5) improved the activity of autologous NKAE cells by 10% (96.4% ± 19%) leading to a nearly complete destruction of U-266 MM cells, and that of CD45RA- allogenic healthy cells in 19% (47.4% ± 12.6%). Nevertheless, CD45RA- autologous T cells transduced with NKG2D-CAR minimally improved their activity by 5.8% (22.5% ± 10.6%). Additionally, the CAR-NKAE cells were able to destroy the clonogenic tumor cell responsible for the progression of the MM from RPMI-8226 cell line. At an 8:1 ratio the CAR-NKAE cells were able to destroy 71.2% ± 2.5% of the clonogenic tumor cells, while the NKAE reached 56.5% ± 2.6% at a maximum ratio of 32: 1. The toxicity of the CAR-NKAE cells on healthy tissue from the same patient was assessed, and no activity against autologous PBMCs was observed, 1,8% at a maximun ratio of 32:1 (effector:target).
In vivo. NKAE cells and CAR-NKAE cells were efficient in abrogating MM growth. However, CAR-NKAE cells treatment showed higher efficiency 14 days after tumor cells injection. Forty-two days after tumor cells injection, only animals receiving CAR-NKAE cells treatment remain free of disease (Figure 1).
Conclusions
It is feasible to modify primary NKAE cells and CD45RA- T cells from primary MM cells to safely express an NKG2D-CAR. Our data show that CD45RA- T cells from patients are not effective in vitro against MM even once transduced with our CAR. The resulting CAR-NKG2D NKAE cells are the most appropriate strategy for the destruction of MM in vitro and in vivo in our model. These results form the basis for the development of an NKG2D-CAR NK cell therapy in MM.
Rio:Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Martinez-Lopez:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Vivia: Honoraria; Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding.
Introduction
Mitochondria controls crucial biological pathways such as proliferation, apoptosis and cell growth. However, the implication of mitochondrial activity in the pathogenesis of Multiple ...Myeloma (MM) still remains unknown and only a few studies connect the mitochondrial status and MM.
We planned to decipher the role of the mitochondria in the MM mechanism of resistance and the potential exploitation of mitochondrial activity as a functional target in the MM therapy.
Methods
In order to understand the role of mitochondria in MM and its therapeutic exploitation, firstly we explored factors involved in the mitochondrial function (c-Myc, HNRNPK, TFAM, NRF1 and EF-Tu) from 770 MM patients RNAseq CoMMpass℠ data. Furthermore, we performed different studies in our MM 77 patients set: gene expression validation by RT-PCR (n=40), protein expression (COXII) by IHC (n=28); and mitochondrial activity (COX activity) by histoenzymatic-HE assay (n=11). Additionally, we analyzed the impact of bortezomib in the mitochondria regulator CD38 in 50 samples (n=30 RVD, n=20 RD regimens), at diagnosis and 6/9 months follow-up MM patients.
We have tested the effect of tigecycline, a mitochondrial inhibitor, in three regimens: monotherapy, pre-treament of tigecycline (48h) with consecutive bortezomib treatment, and in combination with bortezomib in the MM cell lines JJN3, L363 and NCI-H929. To characterize the molecular mechanisms underlying the cytotoxic effect of tigecycline we analysed mitochondria load and activity (MitoTracker green and red) OXPHOS expression by WB and COX2 activity by HE assay. Finally, we followed an in vivo experiment in NSG mice (n=40) engrafted with the JJN3-GFP cell line (1x106) via tail vein and treated by 4 weeks. Analysis of the in vivo imaging and survival curve were obtained.
Results
The higher expression of factors involved in the mitochondrial function such as: c-Myc, HNRNPK, NRF1 and EF-Tu predict MM poor outcomes (Fig.1A). Furthermore, mitochondrial representative gene and protein expression and activity were found increased in MM relapse stage patients. We showed overexpression of C-Myc, TFAM and EF-Tu on the MM relapsed group (Fig. 1B). Moreover, IHC reveals overexpression of mitochondrial COXII protein in relapse MM patients (p-value ** < 0.001) (Fig. 1C). By functional assays we have demonstrated that gene/protein overexpression drives to an increase of activity (COX HE) in MM at relapse (p-value ***< 0.0001). (Fig. 1D). Moreover, we observed an increase of CD38 expression in patients with RVD regimen, but not without bortezomib (RD regimen) (Fig. 1E). Together these results suggest elevation of mitochondrial activity plays a role in the mechanism of resistance to treatment and/or progression of MM and the consequent relapse of the patients.
In vitro studies with tigecyline and bortezomib showed cytotoxic effects in three MM cell lines (IC50 JJN3 11,91 µM; IC50 L363 10,21 µM and NCI-H929 26,37 µM, p-value *< 0.05). Moreover, bortezomib and tigecyline showed high levels of synergism (CI 0,19) (Fig. 1F). In fact, the “conditioning” treatment with tigecyline revert the resistance to bortezomib. The cells treated with tigecycline reflect diminishing in the mitochondria respiration by MitoTracker assays, decrease of COX activity and respiratory chain complexes, suggesting a reduction of mitochondrial activity (Fig. 1G). These molecular effects are exacerbated by the tigecycline and bortezomib combination. However, bortezomib monotherapy not decrease or inclusive, increase, all the molecular mechanisms of mitochondria studied. Finally, mice groups treated with tigecycline alone or in combination with bortezomib reported a better survival and lower JJN3-GFP infiltration (p-value *< 0.05) (Fig. 1H).
Conclusion
To sum up, these findings highlight new vulnerabilities in myeloma cells, suggesting a potential therapeutic target in the treatment of the disease. The metabolic activation of myeloma cells with the collaboration of CD38 and/or c-Myc overexpression or his regulators (e.g. HNRNPK) in response to bortezomib treatment lead an increase of mitochondria respiration. These data confirm the important role of mitochondria in the loss of efficacy in inhibitors of proteasome treatment. Thus, mitochondrial respiration emerges as a novel target in bortezomib relapsed MM patients, and, potentially, in multiple c-Myc, HNRNPK and CD38 overexpression neoplasms.
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Munshi:Adaptive: Consultancy; Oncopep: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Abbvie: Consultancy.
The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide ...(NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-theta (PKC-theta ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of Beta-actin and PKC-theta from the lamellipodium-like distal (d)-SMAC, promoting PKC-theta activation. Furthermore, eNOS-derived NO S-nitrosylated Beta-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-theta was corroborated by overexpression of PFN1- and actin-binding defective mutants of Beta-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-theta at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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![eNOS S-nitrosylates [Beta]-...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/1544-9173/small "eNOS S-nitrosylates [Beta]-actin on Cys374 and regulates PKC-[theta] at the immune synapse by impairing actin binding to profilin-1")