Background: Multiple myeloma is a haematological malignancy whose burden falls predominantly on older adults. Absolute case numbers have risen substantially in England over recent decades, yet the extent to which this reflects changing biological risk versus population ageing and growth has remained unclear. Quantifying the relative contributions of these drivers is essential for anticipating future service demand. Methods: We analysed National Cancer Registration and Analysis Service data from 1995 to 2019 across 106 NHS Integrated Care Boards in England, stratified by age, sex, and geography. Spatial autocorrelation analyses were used to assess geographic clustering in incidence-rate change and absolute case counts. Historical trends were linked to Office for National Statistics cohort-component population projections under twelve demographic scenarios to estimate myeloma case numbers through to 2050. Findings: Absolute myeloma diagnoses approximately doubled between 1995 and 2019, whereas age-specific incidence rates rose only modestly across the same period. No significant geographic clustering in incidence-rate change was detected, whilst absolute case counts were strongly spatially clustered, with increases concentrated in London and the South East. A substantial male excess in incidence widened markedly with age, with rates in males aged 80 and over approaching double those in females. Projections ndicate that annual case numbers may double again by 2050, with growth concentrated almost entirely in the oldest age groups. Interpretation: The expanding myeloma burden in England is driven predominantly by demographic change rather than shifts in underlying biological risk. Future service configuration, workforce planning, and therapeutic development must be aligned to the needs of an older, frailer myeloma population.

Regulation of oncogenic transcriptional programs in multiple myeloma requires the interplay of histone modifications, their writers and readers with lineage-affiliated transcription factors. The transcription factors IKZF1/3, IRF4 and MYC form an aberrant, myeloma-specific regulatory loop that drives myelomagenesis and resistance to immunomodulatory drugs (IMiDs) such as lenalidomide. Chromatin-based mechanisms that regulate these processes remain incompletely understood. Here we investigate the role of CXXC1, a core component of the H3K4 methyltransferase complex COMPASS, in the activity of the IKZF1/3-IRF4-MYC regulatory loop. We find that clinically, high CXXC1 expression is associated with high-risk proliferative, adverse prognosis disease. Consistent with this, CXXC1 is a myeloma dependency and it regulates cellular fitness programs including cell cycle, MYC targets and DNA damage response. High CXXC1 expression in primary myeloma cells is associated with higher chromatin accessibility, while acute depletion of degron-tagged CXXC1 further validates its role in regulating myeloma cell fitness programs and high-risk transcriptional signatures. CXXC1 interacts with and extensively co-binds to chromatin with IRF4 and IKZF3. Notably, in both lenalidomide-sensitive and -resistant myeloma cells, CXXC1 depletion results in loss of IRF4 and IKZF3 chromatin binding and in parallel it 'breaks' the IRF4 transcriptional self-regulatory loop. Thus, CXXC1 and COMPASS emerge as novel therapeutic targets in IMiD-sensitive and -resistant myeloma by regulating the activity of IRF4 and essential myeloma cell fitness programs.

ABSTRACT:Current therapies, including autologous chimeric antigen receptor (CAR) T-cell immunotherapy, fail to cure half of infants with KMT2A-rearranged acute lymphoblastic leukemia (KMT2Ar-ALL), a disease characterized by frequent central nervous system involvement, poor treatment response, early relapse, and lineage switching. More effective treatment strategies, including the availability of off-the-shelf immunotherapies, is particularly relevant in infants. PROM1/CD133 is a direct target of KMT2A-fusion oncoproteins and is expressed on leukemic cells. Allogeneic invariant natural killer T (iNKT) cells, "innately" more powerful effectors than T cells, can be deployed off-the-shelf without risk of acute graft-versus-host disease. Here, we equip iNKT cells with CD19- and/or CD133-targeting CARs, and investigate their antileukemia activity against KMT2Ar-ALL in relevant in vitro and in vivo models. Compared with monospecific counterparts and dual, bispecific CAR T cells, bispecific CD19-CD133 CAR-iNKT cells have a more potent antileukemia activity, effectively targeting both CAR antigen-high and -low leukemia. Bispecific CAR-iNKT cells eradicate medullary and, notably, leptomeningeal leukemia, and induce sustained remissions without discernible hematologic toxicity. Mechanistically, the more potent antileukemia effect of CAR-iNKT cells over CAR T cells is mediated by a pronounced CAR-dependent and CAR antigen-dependent upregulation of the innate activating receptor NKG2D on CAR-iNKT cells, and its engagement by its corresponding ligands on KMT2Ar-ALL cells. This ensures effective leukemia targeting even with downregulation of CD133 or CD19. Thus, by engaging with 2 different types of leukemia-associated antigens, that is, CAR antigens and NKG2D ligands, CAR-iNKT cells provide a powerful platform for the treatment of KMT2Ar-ALL. This approach can be readily adapted for other high-risk malignancies, including those with otherwise difficult to target leptomeningeal involvement.

iNKT cells are emerging as a highly promising immunotherapy platform for the treatment of cancer. To maximise the anti-cancer activity of CAR-iNKT against the blood cancer multiple myeloma we investigated optimal CAR designs and their combination with new iNKT-specific engagers. We find that amongst five different CAR endodomains, underpinned by increased avidity and a cross talk between Plexin D1 on CAR-iNKT and Semaphorin 4 A on myeloma cells, BCMA CD28z CAR-iNKT exert the highest anti-myeloma activity. Notably, CD28z CAR-iNKT outperform their CAR-T counterparts. To expand the anti-myeloma potential of CAR-iNKT, we designed and validated a high efficacy BCMA iNKT-specific engager which exerts significant anti-myeloma activity in conjunction with adoptively transferred iNKT cells. Finally, combined, dual target therapy with FCRL5 CAR-iNKT and BCMA iNKT engagers outperforms FCRL5 CAR-iNKT and limits immune escape of FCRL5-negative myeloma. Thus, optimised iNKT-based, dual-target, dual-modality immunotherapy has enhanced anti-tumor activity against multiple myeloma and potentially other malignancies. iNKT cells are a subset of T cells with both innate and adaptive features, emerging as a promising immunotherapy platform due to their rapid response. Here, the authors develop a CAR-iNKT strategy incorporating a CD28ζ co-stimulatory domain and two bispecific iNKT engagers to enhance multiple myeloma targeting.

Current therapies, including autologous CAR-T immunotherapy, fail to cure half of infants with KMT2A-rearranged acute lymphoblastic leukemia (KMT2Ar-ALL). Here we deploy allogeneic iNKT cells, innately more powerful effectors than T cells, and equip them with CD19- and/or CD133-targeting CARs. Compared to mono-specific counterparts and bi-specific CAR-T, CD19-CD133 bi-specific CAR-iNKT have more potent anti-leukemia activity, they effectively target CAR antigen-low leukemia, eradicate medullary and leptomeningeal leukemia and induce sustained remissions without discernible hematologic toxicity. Mechanistically, dynamic CAR- and CAR antigen-dependent upregulation of the activating innate receptor NKG2D and its engagement by corresponding ligands on KMT2Ar-ALL cells lead to more potent anti-leukemia effect of CAR-iNKT over CAR-T cells, including against CAR antigen-negative leukemia. Thus, by engaging with two different types of leukemia-associated targets, CAR-iNKT provide a powerful platform for the treatment of KMT2Ar-ALL. This approach can be readily adapted for other high-risk malignancies, including those with otherwise difficult to target leptomeningeal involvement. ### Competing Interest Statement AK, TM, AR, HR, NE, BL, CH and R.J.M.B are co-authors of a patent based on the work presented here. AK chairs the scientific advisory board of and holds share options in Arovella Therapeutics. TAM is a shareholder in and consultant for Dark Blue Therapeutics. R.J.M.B.-R. is a co-founder and consultant for Alchemab Therapeutics Ltd, and co-founder of Theraimmune.

Multiple myeloma (MM) is the second most common hematological malignancy. It is marked by widespread transcriptional dysregulation and is highly dependent on key transcription factors such as IRF4, MYC, IKFZ1/3 and the lysine acetyltransferases P300/CBP. Several therapeutic approaches target these factors, including immunomodulatory drugs (IMiDs), which degrade IKZF1/3 to downregulate IRF4 and MYC. Despite advances in treatment strategies, relapse is inevitable. Moreover, relapsed patients become refractory to their previous treatments; resistance has been demonstrated to arise through genetic and epigenetic mechanisms. This highlights the importance of developing new myeloma drugs to better treat relapse/refractory patients. Inobrodib is a P300/CBP bromodomain inhibitor that is currently in a phase I/IIa clinical trial for relapsed/refractory MM (NCT04068597). Initial findings indicate that pomalidomide-refractory patients are resensitized when co-treated with inobrodib. Aside from its favorable therapeutic profile, it is also well-tolerated in patients. Its success appears to pivot on its ability to downregulate IRF4 and MYC, causing cell cycle arrest in MM cell lines. Considering the incurable nature of MM, we hypothesized that patients successfully treated with inobrodib will inevitably relapse and become refractory. Hence, we aimed to model inobrodib resistance mechanisms and identify novel vulnerabilities that may be harnessed as potential treatments for inobrodib-resistant patients, prior to their emergence in clinic. We evolved inobrodib resistance in three MM cell lines (KMS12, H929 and JJN3) representing the most common translocation subtypes [t(11;14), t(4;14) and t(14;16), respectively] and obtained clonal populations for analysis. These cells also demonstrated resistance against the acetyltransferase inhibitor A485, indicating a general loss of dependence on P300/CBP. We captured transcriptional and epigenetic changes in response to inobrodib through RNA-seq, ChIP-seq and ATAC-seq in sensitive and resistant cell lines to elucidate resistance mechanisms. We observed widespread transcriptional differences between sensitive and resistant cells, along with genome-wide redistribution of H3K27ac, P300 and IRF4 binding, and differential responses to inobrodib treatment. By comparing clonal populations of resistant cells, we found evidence of at least four distinct resistance pathways. However, maintenance of IRF4 expression following inobrodib treatment was a common feature in all resistant clones. Surprisingly, despite no previous exposure to IMiDs, the inobrodib-resistant cells also displayed resistance towards pomalidomide. However, combination treatment with pomalidomide and inobrodib was able to drive IRF4 downregulation, resensitizing inobrodib-resistant cells to treatment. Confirming the importance of IRF4 in resistance, we found that exogenous IRF4 expression in sensitive cell lines increased their tolerance to both inobrodib and pomalidomide. Together, these findings implicate IRF4 as a common driver of inobrodib and IMiD resistance. Despite identifying multiple pathways to inobrodib resistance, we found that IRF4 maintenance in the presence of inobrodib is a shared feature. This ability likely explains inobrodib and IMiD cross-resistance in these cells. Given that both pomalidomide and inobrodib act via IRF4 downregulation, combination treatment appears to be an effective strategy to target patients refractory against either drug. Additionally, we propose that direct targeting of IRF4 activity may be a fruitful avenue for further investigation where IMiD and inobrodib treatment is unsuccessful.

Invariant natural killer T (iNKT) cells are rare innate lymphocytes that bridge innate with adaptive immunity. Their invariant T cell receptor (iTCR) recognises lipid antigens presented by CD1d, a nonpolymorphic MHC class I-like glycoprotein. iNKT cells have a protective effect against acute-graft-versus-host-disease (aGVHD) and they can be deployed as a powerful off-the-shelf CAR immunotherapy platform. Emerging pre-clinical evidence shows that CAR-iNKT cells outperform CAR-T against blood cancers, including B-cell acute lymphoblastic leukemia (B-ALL). Our previous transcriptome analysis showed higher expression of ITGA4 (alpha-4) and ITGB1 (beta-1), the constituent chains of VLA-4 by iNKT, compared to same donor T cells. VLA-4 mediates immune cell adhesion to endothelial cells via binding to vascular cell adhesion molecule 1 (VCAM-1), a pre-requisite for immune cell trafficking across the blood brain barrier (BBB), migration across the choroid plexus epithelium and entry into the central nervous system via the cerebrospinal fluid. Since B-ALL is associated with high incidence of leptomeningeal leukemia, we hypothesised that CAR-iNKT would be more effective in trafficking into the leptomeningeal space and limiting leptomeningeal leukemia than CAR-T cells. To address this, we investigated the expression of VLA-4 in resting and CD3/CD28 antibody-stimulated healthy donor T and iNKT cells. We observed that while 86.99% of resting iNKT cells highly co-expressed the two subunits of VLA-4, only 29.85% of T cells had the same level of co-expression (n=10, p < 0.0001). iNKT cells were also found to bind significantly more soluble VCAM-1 than T cells, assessed by flow cytometry, (n=5, p < 0.01) suggesting higher conformationally active VLA-4 expression in iNKT cells. Next, we evaluated VLA-4 expression and activity on CAR-iNKT and CAR-T cells using CARs targeting B-ALL (CD19-CD133 bispecific CAR) or multiple myeloma (BCMA CAR). First, we compared the cytotoxic activity of same donor CAR-T, CAR-iNKT cells and same donor non-transduced (NT) T and iNKT cells against CD19/CD133 (SEM) and BCMA (MM1.S)-expressing cell lines. CAR-iNKT showed significantly higher cytotoxicity of targets than same donor CAR-T cells, with very limited non-specific killing observed by NT cells. CAR-T and CAR-iNKT cells had the same levels of beta-1 expression, however only 30% of CAR-T cells exhibited similar level of alpha-4 expression to CAR-INKT cells (n=5, p < 0.01). To assess the functional role of VLA-4 in CAR-T and CAR-iNKT cells, we measured their adhesion capacity on immobilized soluble VCAM-1 and VCAM-1-expressing HUVEC and BBB-derived hCMEC/D3 endothelial cells, under static and shear stress conditions, either in the presence of natalizumab, an alpha-4 blocking antibody, or its IgG4 isotype. Both CAR- and NT-iNKT cells showed significantly higher static adhesion than their T cells counterparts, with CAR-iNKT cells exhibiting the highest overall adhesion capacity under flow (n=5, p < 0.001), followed by NT-iNKT cells (n=5, p < 0.01); these differences were sustained at the highest shear stress level. Natalizumab blocking significantly decreased cell adhesion, less so in iNKT cells, consistent with the higher expression levels of conformationally active VLA-4 compared to T cells. These findings highlight iNKT cells' inherent ability for firmer cell adhesion than T cells, in a VLA-4-dependent manner. To test our hypothesis in vivo, we employed a luciferase-expressing SEM B-ALL xenograft model in NSG mice. Treatment on day 17, when leukemia cells are readily identified in the meningeal space, showed that 107CD19/CD133 CAR-iNKT cells were more effective, compared to same dose CAR T cells, in reducing leukemia burden in the bone marrow, spleen and in near elimination of leukaemia cells in the meninges at 24 hours and even more profoundly at 48 hours post treatment. We conclude that CAR-iNKT cells are inherently better poised than CAR-T cells for treating high-burden pre-clinical meningeal leukemia, in part due to their higher, VLA-4-mediated ability to adhere to endothelial cells of the BBB and increased rentention in the leptomeningeal space. These findings have wider implications for cellular immunotherapy of blood and solid tumor cancers affecting the brain and the meninges.

Multiple myeloma (MM) and its aggressive subtype plasma cell leukemia (PCL) are transcriptionally-driven hematological malignancies, heavily dependent on enhancer activity. Enhancers are distal regulatory elements at which transcription factors (TFs) bind, recruiting coactivators that promote chromatin remodeling to stimulate target gene expression via physical proximity of enhancer and promoter. MM cells show high expression of essential TFs, including IRF4, MYC, PRDM1 and IKZF1/3, which function by binding to and activating oncogenic enhancers. A detailed understanding of chromatin regulation in MM patients requires the comparison of histone modifications, chromatin accessibility and TF/cofactor binding distribution within the same cells, which has so far been unachievable. Patient analysis has mainly relied on ATAC-seq and RNA-seq, with limited inference of TF binding and chromatin state, owing to the difficulty in obtaining the large numbers of MM cells required for more detailed characterization. Many observations of enhancer function in MM therefore come from cell lines, which only offer an approximation of MM physiology. To overcome the limitations of in vitro models and primary cell availability, we employed TOPmentation (TF-OPtimized ChIPmentation) to generate ChIP-seq-quality data for CD138+ cells from MM and PCL patients. This technique uses as few as 100,000 cells for histone modifications, and 250,000 cells for TFs, allowing us to profile up to 15 features per patient. In addition, we used the base-pair resolution chromosome conformation capture technique micro-capture-C (MCC) to visualize enhancer-promoter interactions in a PCL patient sample. This allowed us to dissect large oncogenic enhancers, identifying the key TF binding sites within each enhancer that directly contact the gene promoter. Using this dual approach, for the first time we have generated integrated maps of TF occupancy, active and repressive histone modifications, and chromatin interactions in MM patient samples. We characterized the epigenetic landscapes of two t(11;14) and two t(4;14) patients, finding broadly similar chromatin profiles in each subtype. Key MM enhancers were retained at oncogenes including IRF4, MYC,PRDM1 and IKZF1/3, indicating a convergence of gene regulation in MM originating from distinct initiating genetic events. At many enhancers, we observed multiple TFs co-binding at the same sites, indicating cooperative activation of target genes. By combining TOPmentation with MCC, we found that many of these sites interact with target gene promoters, directly implicating them in gene regulation. In MM, patients typically display mutually exclusive upregulation of CCND1 or CCND2. As previously established, we found strong activation at the CCND1 locus by IGH translocation in t(11;14) patient samples, whereas in t(4;14) patients CCND2 was upregulated via an upstream super-enhancer. Surprisingly, in each subtype the silent CCND gene was not actively repressed, but rather existed in a poised, bivalent state marked by active H3K4me3 and repressive H3K27me3 modifications. This argues that intricate regulation of CCND1/2 is required to maintain optimal levels of expression for tumor growth. Comparison of patient and cell line data showed broad conservation of epigenetic features. However, we also observed examples of distinct gene regulation, for example at the PRDM1 locus. In several MM cell lines (including KMS12, H929 and MM1S), transcription of PRDM1 initiates from a distal promoter. However, this locus was inactive in the patient samples analyzed, and instead an alternate, proximal promoter was favored. This study for the first time reports the genomic binding distribution of TFs and histone modifications in MM patient cells, demonstrating the importance of comprehensive epigenomic analysis to capture the complexity in MM gene regulation. This emphasizes the need for validation of cell line models to provide a more physiologically relevant understanding of enhancer-driven oncogene regulation. As we expand this patient-derived dataset, it will provide a valuable resource to better understand MM biology and inform future strategies for precision epigenetic therapy.

iNKT cells are emerging as a highly promising immunotherapy platform for the treatment of cancer. To maximise the anti-cancer activity of CAR-iNKT against the blood cancer multiple myeloma we investigated optimal CAR designs and their combination with novel iNKT-specific engagers. We find that amongst five different CAR endodomains, underpinned by increased avidity and a cross talk between Plexin D1 on CAR-iNKT and Semaphorin 4A on myeloma cells, BCMA CD28z CAR-iNKT exert the highest anti-myeloma activity. Notably, CD28z CAR-iNKT outperform their CAR-T counterparts. To expand the anti-myeloma potential of CAR-iNKT, we designed and validated a high efficacy BCMA iNKT-specific engager which exerts significant anti-myeloma activity in conjunction with adoptively transferred iNKT cells. Finally, combined, dual target therapy with FCRL5 CAR-iNKT and BCMA iNKT engagers outperforms FCRL5 CAR-iNKT and limits immune escape of FCRL5-negative myeloma. Thus, optimised iNKT-based, dual-target, dual-modality immunotherapy has enhanced anti-tumor activity against multiple myeloma and potentially other malignancies. ![Figure][1] ### Competing Interest Statement AK and KP report holding of options/shares in Arovella Therapeutics Ltd. Kay Kendall Leukaemia Fund, https://ror.org/03j2wfg84, KKL1360 Blood Cancer UK, https://ror.org/0055acf80, 24013 MRC UKRI-AstraZeneca Fellowship, MR/X004600/1 Cancer Research UK, DRCPGM\100058 Imperial College London UKRI Impact Acceleration Account, MR/X502959 [1]: pending:yes

IRF4 is a plasma cell lineage-defining transcription factor that orchestrates important aspects of the oncogenic transcriptome in multiple myeloma (MM) largely through the deregulated IKZF1/3-IRF4-MYC axis. This transcription factor feed-forward loop is also critical for response and resistance to immunomodulatory drugs (IMiDs), a cornerstone of MM treatment. IMiD-induced degradation of IKZF1 and IKZF3 leads to reduced expression of IRF4 and MYC and myeloma cell death. Conversely, resistance to IMiDs, often due to impaired IKZF1/IKZF3 degradation, converges to sustained IRF4 expression and transcriptional activity. Therefore, a more in-depth understanding of the chromatin-based mechanisms of IRF4 activity in myeloma cells would inform its direct or indirect therapeutic targeting in both IMiD-sensitive and -resistant MM. We previously found that the chromatin factor CXXC1 is most highly expressed in myeloma cells as compared to other hematologic and solid tumor cells while chromatin accessibility followed by footprinting analysis suggested higher frequency of CXXC1 chromatin binding in myeloma as compared to normal plasma cells. CXXC1 binds unmethylated CpG islands and is part of the COMPASS complex that catalyses histone H3K4me3, a histone mark linked to transcriptional activation. Based on these observations, we investigated the hypothesis that CXXC1 regulates important oncogenic transcriptional programmes in MM. We found that in different myeloma cells, including the MAF-translocated MM1.S myeloma cell line, shRNA-mediated CXXC1 knock down or degron-mediated CXXC1 acute protein depletion are toxic to myeloma cells. Transcriptome analysis of CXXC1-depleted MM1.S cells followed by pathway enrichment analysis and relevant cellular assays suggest that CXXC1 activates gene programs required for cell proliferation, glycolytic metabolism, unfolded protein response and DNA damage response. Proteomics analysis involving immunoprecipitation of CXXC1 followed by mass spectrometry in MM1.S cells confirmed association of CXXC1 with several components of the COMPASS complex, including the SETD1A/B histone methyltransferases. Notably, it also identified IRF4 and IKZF3 (Aiolos) as direct CXXC1 interactors. Consistent with these findings, ChIP-seq in MM1.S and primary myeloma cells showed extensive co-binding of CXXC1 with IRF4, IKZF3 and IKZF1 at promoters and enhancers of key genes, including MYC. ChIP-seq after degron-induced depletion of CXXC1 showed drastic reduction of IRF4 chromatin binding, correlating with reduced CXXC1 occupancy. No changes in long range chromatin interactions were observed by Hi-ChIP assay upon CXXC1 depletion, suggesting that CXXC1 is not required for 3D chromatin organisation. Further epigenetic profiling showed that while H3K27ac abundance was preserved, there was a notable contraction of the size and/or reduction of the intensity of signal of H3K4me3 broad domains, previously linked to transcriptional activation of lineage identity genes. Indeed, such broad domain changes were found at the regulatory areas of several plasma cell lineage-defining genes such as IRF4, PRDM1 and XBP1. To explore the role of CXXC1 in IMiDs resistance, we generated two independent lenalidomide (Len) resistant lines derived from the MM1S CXXC1-degron cells. As expected, upon Len treatment, IRF4 and MYC expression as assessed by western blotting decreased in Len-sensitive cells but was sustained in Len-resistant cells . Nevertheless, degron-mediated CXXC1 depletion was equally toxic to both Len-sensitive and -resistant cells lines and was associated with and extensive loss of IRF4 as well as CXXC1 chromatin binding in both Len-sensitive and Len-resistant cells. Transcriptome analysis of the same cells showed significant downregulation of IRF4 and MYC and of genes involved in mTORC1 signalling, MYC targets, cell cycle, DNA damage and unfolded protein response . We conclude that in myeloma cells, CXXC1 is required for IRF4 binding to chromatin and their co-binding regulates essential, oncogenic transcriptional programmes including expression of IRF4 itself. This dependency of IRF4 chromatin binding on CXXC1 extends to include Len resistant myeloma cells thus providing proof-of-principle that CXXC1, and likely the COMPASS complex, constitute a novel therapeutic target for Len-sensitive and resistant MM.

Abstract Invariant Natural Killer T (iNKT) cells are a unique subset of innate lymphocytes, constituting <1% of human T cells. iNKT cells express a semi-invariant TCR (iTCR) recognizing glycolipids presented by the monomorphic, MHC-like molecule CD1d. Previous studies indicate that due to distinctive TCR constitution and antigen recognition properties, iNKT cells do not induce acute graft-versus-host disease. Based on the CD4 and CD8 expression, mature human iNKT cells can be classified into CD4+CD8-, CD4-CD8- & CD4-CD8+ subsets with overlapping and distinct functions. The functional profiling of CAR19-iNKT cell subsets serves the dual purpose of ensuring their safety and efficacy as a therapeutic intervention. Here we characterized the phenotypic and functional profile of CD4+ and CD4- CAR19-iNKT cells. iNKT cells were isolated with >99% purity from healthy donors' peripheral blood and were transduced with a CD19 CAR-encoding 3rd generation lentivirus. After expansion, the CAR19-iNKT cells were cryopreserved and later analyzed post-thawing. Differential expression of CD27 and PD1 was noted in CAR+CD4+ and CAR+CD4- iNKT cells compared to other markers. For functional profiling, CAR+CD4+ iNKT cells were positively selected, and 24h cytotoxicity assays were performed using several tumor cell lines including SEM, Ramos and C1R-CD1d (+/- α-GalCer). The CAR+CD4- iNKT cells exhibited superior cytotoxicity to multiple tumor cell lines compared to that of CAR+CD4+ iNKT cells from two donors. However, both subsets lysed α-GalCer pulsed C1R-CD1d cells in a comparable manner indicating the potential of these subsets to recognize antigen through the iTCR. Subsequently, we assessed the proliferation ability of the CAR+CD4+ and CAR+CD4- subsets by exposing the cells to three rounds of stimulation, 24h apart, using SEM (CD19+CD1d-) or K562 (CD19-CD1d-) tumor cells with or without IL-15. Proliferation was assessed seven days following the first stimulation. The CAR+CD4+ subset of CAR19-iNKT cells demonstrated faster proliferation than CAR+CD4- cells in the presence of IL-15 after repeated exposure to tumor cells. Ongoing RNA seq analysis of CAR+CD4+/CAR+CD4- cells will further elucidate differences between these subsets. The outcomes of these studies have shown encouraging results, indicating the potential benefit of having diverse subsets among CAR19-iNKT cells for treating CD19+ cancers. The therapeutic potential of CAR+CD4+ iNKT cell subset could be enhanced to match that of CAR+CD4- subset with the use of α-GalCer. In summary, inclusion of both CD4+ and CD4- iNKT cells is critical for the functionality of allogeneic CAR-iNKT cell therapy. Extensive characterization is crucial for clinical translation, but additional research, including preclinical and clinical trials, is essential to determine the safety and effectiveness of CAR19-iNKT cell therapy in real-world applications. Citation Format: Kanagaraju Ponnusamy, Simon Poon, Nicole van der Weerden, Robson Dossa, Michael J. Baker, Mini Bharathan, Anastasios Karadimitris. Allogeneic CD19-directed CAR-iNKT cells and their phenotypic subsets for the treatment of CD19+ hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1332.

BackgroundAcute myeloid leukemia (AML) is the most common malignant myeloid disorder in adults and the fifth most common malignancy in children, necessitating advanced technologies for outcome prediction.MethodThis study aims to enhance prognostic capabilities in AML by integrating multi-omics data, especially gene expression and methylation, through network-based feature selection methodologies. By employing artificial intelligence and network analysis, we are exploring different methods to build a machine learning model for predicting AML patient survival. We evaluate the effectiveness of combining omics data, identify the most informative method for network integration and compare the performance with standard feature selection methods.ResultsOur findings demonstrate that integrating gene expression and methylation data significantly improves prediction accuracy compared to single omics data. Among network integration methods, our study identifies the best approach that improves informative feature selection for predicting patient outcomes in AML. Comparative analyses demonstrate the superior performance of the proposed network-based methods over standard techniques.ConclusionsThis research presents an innovative and robust methodology for building a survival prediction model tailored to AML patients. By leveraging multilayer network analysis for feature selection, our approach contributes to improving the understanding and prognostic capabilities in AML and laying the foundation for more effective personalized therapeutic interventions in the future.

The emergence of multi-omic single-cell technologies over the last decade has led to improved insights into both normal hematopoiesis and its perturbation in a variety of hematological disorders. Diamond-Blackfan anemia (DBA) is one such disorder where single-cell assays have helped to delineate the cellular and molecular defects underlying the disease. DBA is caused by heterozygous loss of function germline variants in genes encoding ribosomal proteins (RPs). Despite the widespread role of ribosomes in hematopoiesis, the most frequent and severe cytopenia in DBA is anemia. In this review we will discuss how single cell studies- including clonogenic cell culture assays, fluorescence activated cell sorting (FACS) and single cell RNA sequencing (scRNAseq)-have led to insights into the pathogenesis of DBA. The main therapies are regular blood transfusions, glucocorticoids or hematopoietic stem cell transplantation (HSCT) but all are associated with significant morbidity and mortality. We will therefore outline how single-cell studies can inform new therapies for DBA. Furthermore, we will discuss how DBA serves as a useful model for understanding normal erythropoiesis in terms of its cellular hierarchy, molecular regulation during homeostasis and in response to ‘stress’.

Abstract Purpose: Molibresib is a selective, small molecule inhibitor of the bromodomain and extra-terminal (BET) protein family. This was an open-label, two-part, Phase I/II study investigating molibresib monotherapy for the treatment of hematological malignancies (NCT01943851). Patients and Methods: Part 1 (dose escalation) determined the recommended Phase 2 dose (RP2D) of molibresib in patients with acute myeloid leukemia (AML), Non–Hodgkin lymphoma (NHL), or multiple myeloma. Part 2 (dose expansion) investigated the safety and efficacy of molibresib at the RP2D in patients with relapsed/refractory myelodysplastic syndrome (MDS; as well as AML evolved from antecedent MDS) or cutaneous T-cell lymphoma (CTCL). The primary endpoint in Part 1 was safety and the primary endpoint in Part 2 was objective response rate (ORR). Results: There were 111 patients enrolled (87 in Part 1, 24 in Part 2). Molibresib RP2Ds of 75 mg daily (for MDS) and 60 mg daily (for CTCL) were selected. Most common Grade 3+ adverse events included thrombocytopenia (37%), anemia (15%), and febrile neutropenia (15%). Six patients achieved complete responses [3 in Part 1 (2 AML, 1 NHL), 3 in Part 2 (MDS)], and 7 patients achieved partial responses [6 in Part 1 (4 AML, 2 NHL), 1 in Part 2 (MDS)]. The ORRs for Part 1, Part 2, and the total study population were 10% [95% confidence interval (CI), 4.8–18.7], 25% (95% CI, 7.3–52.4), and 13% (95% CI, 6.9–20.6), respectively. Conclusions: While antitumor activity was observed with molibresib, use was limited by gastrointestinal and thrombocytopenia toxicities. Investigations of molibresib as part of combination regimens may be warranted.

Introduction Most T cell receptor (TCR)Vβ chain-expressing T cell lymphomas (TCL) including those caused by Human T cell leukaemia virus type-1 (HTLV-1) have poor prognosis. We hypothesised that chimeric antigen receptor (CAR)-mediated targeting of the clonal, lymphoma-associated TCRβ chains would comprise an effective cell therapy for TCL that would minimally impact the physiological TCR repertoire. Methods As proof of concept, we generated CAR constructs to target four TCRVβ subunits. Efficacy of the CAR constructs was tested using conventional T cells as effectors (CAR-T). Since invariant NKT (iNKT) cell do not incite acute graft-versus-host disease and are suitable for ‘off-the-shelf’ immunotherapy, we generated anti-TCRVβ CAR-iNKT cells. Results We show that anti-TCRVβ CAR-T cells selectively kill their cognate tumour targets while leaving >90% of the physiological TCR repertoire intact. CAR-iNKT cells inhibited the growth of TCL in vivo, and were also selectively active against malignant cells from Adult T cell leukaemia/lymphoma patients without activating expression of HTLV-1. Discussion Thus we provide proof-of-concept for effective and selective anti-TCRVβ CAR-T and -iNKT cell-based therapy of TCL with the latter providing the option for ‘off-the-shelf’ immunotherapy.

Despite the approval of three autologous CAR-T cell products, B-cell malignancies still represent an unmet need as several of the relapsed/refractory patients are ineligible for autologous CART cell therapies. Invariant natural killer T (iNKT) cells are a unique subset of immune cells that display properties of both T cells and natural killer cells and bridge the innate and adaptive immune responses upon antigen recognition. Unlike conventional β T cells, iNKT cells express an invariant T cell receptor (TCR) that recognize glycolipids presented in the context of the monomorphic, MHC-class I related molecule, CD1d. iNKT cells are ideal cell types for allogeneic, off the shelf approach as they can target cancers without the risk of graft-versus-host disease (GvHD), circumventing the need to delete or knock out the endogenous TCR. Here, we report the engineering of ‘off-the-shelf’ iNKT cells directed against CD19 and their robust in vitro and in vivo functional profile. Briefly, peripheral blood derived iNKT cells were isolated from healthy donors and were engineered to express a CD19 CAR using a 3rd generation lentiviral vector. To demonstrate the CAR-dependent and independent anti-tumor activity, CAR19-iNKT cells were compared in vitro against non-transduced (NT) iNKT cells in cytotoxicity assays and intracellular cytokine staining assays. Allogeneic CAR19-iNKT cells upregulated activation markers, secreted cytokines, and lysed cancer cell lines and primary tumor cells from chronic lymphocytic leukemia and marginal zone lymphoma patients in an antigen-specific manner. Excellent post thaw recovery and viability was noted in cryopreserved CAR19-iNKT cells. Finally, the anti-tumor activity of cryopreserved CAR19-iNKT cells were evaluated in an established aggressive NSG mice model of SEM-luc, a B cell lymphoblastic leukemia cell line expressing luciferase. Robust antitumor activity by CAR19-iNKT cells was observed without any obvious adverse effects compared to controls (untreated and NT iNKT cells). The persistence of CAR19-iNKT cells in this model is currently being evaluated. These data demonstrate a unique, off the shelf CAR19-iNKT cell platform for targeting hematological malignancies that combines the specificity of CD19 targeting CAR with the innate features of iNKT cells. The data indicates that iNKT cells are a potent cell type for generating allogeneic off the shelf products for treating a broad spectrum of hematological malignancies and solid tumors by targeting different receptors on the respective tumors. Citation Format: Kanagaraju Ponnusamy, Janani Kanagaraju, Yingxin Wang, Bryan Lye, Hongwei Ren, Sandhya Buchanan, Michael J. Baker, Mini Bharathan, Anastasios Karadimitris. Engineering allogeneic off-the-shelf CD19-directed CAR-iNKT cells without additional genetic manipulations for the treatment of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 913.

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure syndrome, usually caused by loss-of function variants in genes encoding ribosomal proteins. The hallmarks of DBA are anemia, congenital anomalies and cancer predisposition. Although DBA usually presents in childhood, the prevalence in later life is increasing due to an expanding repertoire of implicated genes, improvements in genetic diagnosis and increasing life expectancy. Adult patients uniquely suffer the manifestations of end-organ damage caused by the disease and its treatment, and transition to adulthood poses specific issues in disease management. To standardize and optimize care for this rare disease, in this review we provide updated guidance on the diagnosis and management of DBA, with a specific focus on older adolescents and adults. Recommendations are based upon published literature and our pooled clinical experience from three centres in the United Kingdom (U·K.). Uniquely we have also solicited and incorporated the views of affected families, represented by the independent patient organization, DBA U.K.

iNKT are rare, CD1d-restricted, glycolipid-reactive T cells that bridge adaptive and innate immunity. iNKT cells are characterised by an invariant TCRVa24Ja18 chain nearly always pairing with a TCRVb11 chain (iTCR). The ability of allogeneic iNKT to protect from aGVHD makes them ideal candidates for ‘off-the-shelf’ immunotherapy without need for TCR deletion. Indeed, CAR-iNKT show promise in pre-clinical models of blood cancer as well as early clinical development against B cell lymphoma and outperform CAR-T counterparts. Here we aimed to enhance the modularity of adoptive iNKT cell immunotherapy for blood cancers by designing a bispecific iNKT cell engager (biNTe) in which one arm would engage the iTCR while the other arm would engage tumour antigen(s) of interest. As a proof-of-principle we designed and synthesised a mono- and a bi-valent biNTe, one each, against the clinically validated myeloma target BCMA with purities of 90% and 70% respectively. We focused on the bivalent biNTe which we first used to demonstrate its iNKT cell binding specificity. Indeed, in flow-cytometry assays, biNTe followed by fluorescent anti-human Fc Ab, stained purified iNKT and but not T cells while in the presence of the same iTCR-binding mAb used to generate the biNTes, binding of the bivalent biNTe on iNKT cells was abrogated. This shows the iTCR engaging specificity of the bivalent biNTe. To demonstrate BCMA specificity of the other arm of the biNTe, we used it to stain iNKT cells, followed by staining with biotinylated soluble BCMA and fluorescently labelled streptavidin. This, compared to staining without soluble BCMA, showed strong staining of iNKT cells thus validating the BCMA specificity of the bivalent biNTe. Next, we set up co-cultures of pre-expanded iNKT against the BCMA-expressing and -knockout myeloma cell line MM1.S in the presence of varying concentrations of the biNTe. While, as assessed by a flow-cytometric cytotoxicity assay, only background killing of myeloma cells was observed in the absence of biNTe or when BCMA ko cell were used as targets, in the presence of biNTe, BCMA+ MM1S cells were killed, in a dose dependent manner (Figure 1A). Consistent with iNKT specificity of the biNTe similar co-cultures involving T cells showed only background killing of MM1S cells (Figure 1B). The same results were obtained using the BCMA-expressing myeloma cell line H929 and its corresponding BCMA knockout. The overall estimated EC50 for MM1.S and H929 cells were 6.56 and 3.4 nM respectively. Using the EC50 concentrations we show that biNTe very effectively kill and almost eliminate patient-derived bone marrow myeloma plasma cells (n=3). To test whether engagement of the iTCR by biNTe would impact its ability to engage with CD1d, we co-cultured iNKT and MM1.S cells transduced with CD1D in the presence of biNTe and the activating, iNKT-specific, CD1d-binding glycolipid ligand a-galactosylceramide (aGalCer). We found that compared to no biNTe control and at biNTE concentrations lower than the EC50, killing of myeloma targets cells was consistently increased. This suggests that iTCR retains its ability to engage CD1d and the efficacy of future biNTe-based immunotherapy could be enhanced by aGalCer. While in vivo validation experiments are ongoing, we conclude that biNTe constitute a novel therapeutic approach with notable pre-clinical anti-myeloma activity. We envisage that they can be used in conjunction with other therapeutic modalities such as CAR and iTCR to enhance the modularity and the efficacy of iNKT cells as an ‘off -the-shelf’ platform for the treatment of myeloma, other blood or solid tumour cancers.