Primary Patient Material Research Articles

Paediatric bone marrow mesenchymal stem cells support acute myeloid leukaemia cell survival and enhance chemoresistance via contact-independent mechanism.

Children diagnosed with acute myeloid leukaemia (paediatric AML [pAML]) have limited treatment options and relapse rates due to chemoresistance and refractory disease are over 30%. Current treatment is cytotoxic and in itself has long-lasting harsh side effects. New, less toxic treatments are needed. The bone marrow microenvironment provides chemoprotection to leukaemic cells through cell communication and interaction with mesenchymal stem cells (MSCs), but this is not well defined in pAML. Using primary patient material, we identify a cell contact-independent mechanism of MSC-mediated chemoprotection involving extrinsic soluble factors that is abrogated through inhibition of the JAK/STAT and ERK pathways.

Advancing CAR-T Therapy in Acute Lymphoblastic Leukemia: Multi-Omic Analyses of CD19-Directed CAR-T Cells Enabled By an Ex Vivo Co-Culture Platform

Background: Acute Lymphoblastic Leukemia (ALL) has witnessed remarkable progress in the treatment of patients with Chimeric Antigen Receptor T-cell (CAR-T) therapies, leading to high response rates and durable remissions. However, challenges persist for patients who do not respond to CAR-T treatment, experience relapse, or encounter adverse events such as cytokine release syndrome. Overcoming these challenges necessitates a deeper understanding of mechanisms and the development of advanced technologies and assays. This study focuses on harnessing the potential of an innovative microfluidic platform for high-throughput cell-based assays, enabling multi-omics and functional analysis using a minimal amount of primary patient cells. By utilizing this platform, we aim to gain insight into the dynamics of primary patient material by evaluating responses to aid in selection of promising CAR-T enhancements or combination therapies designed to overcome mechanisms of CAR-T failure or relapse to improve clinical outcomes. Methods: For the cytotoxicity assay, we utilized autologous or allogeneic pairs of CD19-directed CAR-T cells and T-cell negative fractions from ALL patients' blood apheresis samples, containing CD19-expressing cancer cells. A negative control was established using untransduced T-cells. To ensure high cell viability and purity, the cells underwent careful processing and were labeled with long-lived dyes for tracking. Multiple cell suspensions with precise Effector to Target to Negative Fraction ratios were prepared. Only a few thousand cells from each coculture suspension were seeded in microfluidic culture chambers, which required no fluidic control system. Experiments were conducted for up to 96 hours with media replacement every 24 hours. At each endpoint, media was collected for cytokine quantification, and cells were stained for live/dead counting as well as quantification of intracellular or surface marker expression by imaging. After image acquisition, cells were collected for RNA extraction and gene expression analysis. Results: Results demonstrated that the developed co-culture platform allows precise control over culture conditions, ensuring culture of accurate ratios of highly complex cell populations. The viability of primary cells in the device guaranteed functional relevance of the cytotoxicity assays for up to a 96-hour timepoint. Strong CAR-T-mediated cell killing was observed, with a dose-dependent effect depending on the co-culture's relative CAR-T content. Less than 50% cell survival was observed in cocultures with Effector to Target ratio 1:1, as compared to the untransduced T-cell control. Furthermore, strong CAR-T activation was observed with over 60% of CART expressing CD25 and CD69 surface markers, as measured by imaging. A multi-color staining panel allowed for precise monitoring of changes over time within the assay of immune cell populations, including cancer cells, monocytes/macrophages, and NK cells, among others. Cytokine analysis revealed an increased secretion of IL-2, TNFa, IFNg, and Granzyme B in coculture with CAR-T cells compared to untransduced T-cells, with a dose-dependent effect based on the CAR-T cell ratio. Finally, RNA isolated from remaining cells at the end of the assay was of high quality with yields sufficient to enable transcriptome analysis for each treatment condition. Discussion: This study contributes to the development of innovative multiplexed high throughput strategies for in vitro functionality assessments in cell therapy, including combination studies and deeper mechanistic understandings. The platform's ability to generate fully integrated, multi-omics readings from complex allogeneic and autologous cocultures of CAR-T and patient cells offers a new frontier in early discovery and translational cancer research. This opens new possibilities for drug discovery, drug screening, and disease modeling, ultimately advancing our understanding of complex biological processes and accelerating therapeutic development. Future directions include exploring mechanisms of resistance and sensitivity, including donor-specific effector cell functionality, combination studies, impacts of immunosuppressive cell types, patient heterogeneity of target antigen expression or other co-stimulatory molecules, and evaluation of product potency or fitness of incoming apheresis material.

Abstract B045: The effect of metastasis-associated in colon cancer 1 (MACC1) on therapy-induced senescence and its impact on post-senescence-driven metastasis

Abstract Senescence is considered a safeguard program against cancer progression. However, recent studies revealed that these cells, reprogrammed in a senescence-associated fashion to express stemness transcripts, may occasionally re-enter the cell cycle, thereby executing their increased tumorigenicity. Although the effects of senescence, its associated secretome (SASP) and release from senescence on tumor progression have been elucidated before, the role of post-senescent cells in metastasis remains elusive. To enlighten the relationship between senescence, stemness, and metastasis, we employed a key bridging molecule between metastasis and cancer stemness, metastasis-associated in colon cancer 1 (MACC1). Numerous research groups have acquainted its importance as a metastasis inducer, prognostic, and predictive biomarker for more than 20 different tumor entities, including breast and colorectal cancer (CRC). We initially used CRC cell lines with genetically engineered MACC1 expression for our study. Senescence was induced with different chemotherapeutic agents such as 5-FU, mafosfamid (a cyclophosphamide analogue active in vitro), and doxorubicin. Cells with high MACC1 expression were more prone to senesce after treatment. These findings were further validated in CRC patient- and mouse-derived organoids. Across these models, we found MACC1 expression to increase therapy-induced senescence (TIS). Moreover, senescence induction was accompanied by increased MACC1 expression in conjunction with elevated expression of cancer stemness genes like CD44 and LGR5; this increase was more prominently detectable in MACC1 high-expressing cells. Furthermore, senescent cells were forced to exit from senescence through genetic manipulations of independent senescence-essential factors such as overexpression of JMJD2C, which demethylates the senescence-essential H3K9me3 histone mark. Post-senescent cells showed enhanced migration, colony formation, wound healing, proliferation, and increased cancer stemness-related gene expression, especially when compared to never-senescent cells in which we overexpressed JMJD2C prior to chemo-exposure. Our studies were complemented by in vivo work, which showed that metastasis-incapable CRC cells formed metastasis in the liver and lung after their release from senescence. We further used MACC1 inhibitors such as atorvastatin and fluvastatin to interfere with MACC1-induced TIS. The co-treatment of a MACC1 inhibitor with conventional chemotherapeutics reduced senescence entry in cells with high MACC1 expression. These data, consistent with our findings in primary patient material, uncover a hitherto unknown relationship between MACC1 and TIS. Furthermore, senescence release increases the tumorgenicity of the cells, which is more prominent in the cells with high MACC1 expression. In particular, we were able to reduce MACC1-induced senescence by using MACC1 inhibitors, aiming to reduce the senescence release-induced metastasis. Citation Format: Belma Nazli Güllü, Dorothy N. Y. Fan, Dennis Kobelt, Margarita Mokrizkij, Janice Smith, Clemens A. Schmitt, Ulrike Stein. The effect of metastasis-associated in colon cancer 1 (MACC1) on therapy-induced senescence and its impact on post-senescence-driven metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B045.

Dissecting the role of toll-like receptor 7 in pancreatic cancer.

Toll-like receptors (TLRs) are gaining attention for their potential to influence tumor biology both on the level of the tumor cells as well as on the level of the surrounding inflammatory stroma. Previous studies resulted in partly conflicting data on the expression of TLR7 in healthy and neoplastic pancreatic tissues as well as its role in pancreatic tumor biology. We used qRT-PCR and immunohistochemistry to asses TLR7 expression in primary patient material and cell lines. Cell viability was analyzed by MTT assay upon incubation with TLR7 agonist/antagonist. Mouse models were used to investigate the role of TLR7 in vivo. TLR7 is overexpressed in more than 50% of primary human pancreatic ductal adenocarcinoma (PDAC). High TLR7 expression was associated with shorter patient survival, and TLR7 inhibition in cell lines reduced viability in a dose-dependent manner. In contrast, global TLR7 deficiency did not alter survival or overall histopathological tumor features in genetic mouse models of PDAC. TLR7 may have opposing functions in tumor versus stroma cells. Further work is required to more precisely dissect the roles of TLR7 and its ligands in different populations of epithelial and stromal cells and to understand their relative contributions to tumor progression.

23P Enriching for response: Patient selection criteria for A2AR inhibition by EXS-21546 through ex vivo modelling in primary patient material

Novel immunotherapies targeting the adenosine pathway are in clinical trials, however, only modest monotherapy activity has been observed in non-prioritised patient groups. To optimise the chance of success with our A2AR-selective antagonist, EXS-21546 (‘546; NCT04727138, discovered in collab. with Evotec), we work to identify an adenosine-induced immunosuppression biomarker signature for clinical trial patient selection. Here we present initial transcriptional and functional data mapping the adenosine suppressed immune potential at the single cell level, and subsequent modulation through antagonism of A2AR with ‘546, along with first patient-selection modelling to prioritise patients for ‘546 therapy.

Targeting Extracellular Vesicle Secretion in Combination with Venetoclax Synergistically Induces Apoptosis in FLT3-ITD+ AML

Introduction Intrinsic or acquired resistance to apoptosis is a hallmark of human cancers, including different subtypes of acute myeloid leukemia (AML). Among different mechanisms of achieving resistance, disposal of pro-apoptotic factors via extracellular vesicle (EV) release has been described for several cancer models. In AML, RNA and protein trafficking by leukemia-derived EVs has emerged as a mediator of cell-cell communication and a crucial factor in reprogramming the bone marrow (BM) niche into a leukemia permissive microenvironment. In our study, we aimed to characterize the content of primary AML-derived EVs with a special focus on apoptotic factors, and to evaluate the effect of EV release inhibition with FTY720, a FDA approved oral drug for treatment of multiple sclerosis, on apoptosis in combination with venetoclax (VEN) treatment, an inhibitor of the anti-apoptotic protein Bcl-2. Methods BM-derived EVs were purified from healthy donors (n=3), or patients with newly diagnosed AML (FLT3-WT and -ITD, n=10) after written informed consent and approval by the ethics committee in accordance with the Declaration of Helsinki. EVs were characterized by transmission electron microscopy (TEM), western blotting and nano tracking analysis (NTA). Protein and RNA from EVs were isolated and used for proteomic and RNA analyses. The apoptotic response of FLT3-WT (OCI-AML2, OCI-AML3) or FLT3-ITD (MOLM-13, MV4-11) AML cell lines and primary samples to EV release inhibitor FTY720 was assessed by flow cytometry with AnnexinV/PI. FTY720-induced EV release inhibition and intracellular EV accumulation was determined by Western Blot analysis of EV markers and visualized by immunostaining. Finally, the combination treatment of FTY720 with VEN was tested. Results TEM and immunoblotting confirmed a pure vesicle fraction (30-150 nm) with expression of characteristic markers enriched (CD63, CD81, HSP70) or absent (GAPDH, ß-actin) in EVs. NTA showed no differences in EV numbers released by AML compared to healthy cells. However, AML EVs were significantly larger in size. EV content was significantly different comparing healthy to AML-derived EVs and bioinformatic analysis from RNA sequencing revealed enrichment in pathways involved in regulation of apoptosis specifically in AML FLT3-ITD EVs (Figure 1). Of note, hsa-miR-196b-5p target interaction analysis identified BCL-2 as an exemplary anti-apoptotic target gene (Figure 2). Concordantly, AML cell lines harboring FLT3-ITD showed higher sensitivity to the EV release inhibitor FTY720 compared to FLT3-WT (IC50 = 6.2µM, 6.6µM, 13.7µM and 10.2µM respectively for MOLM-13, MV4-11, OCI-AML2 and OCI-AML3). FTY720 treatment induced intracellular EV accumulation and modulated EV marker expression confirming its effect on the EV machinery. Interestingly, FTY720 co-treatment potentiated apoptosis induced by VEN alone in both FLT3-ITD cell lines while FTL3-WT cells were still resistant to VEN and healthy hematopoiesis was unaffected. Conclusions Taken together, these results suggest that FLT3-ITD AML cells establish a protective mechanism against apoptosis by disposing of positive regulators of apoptosis via EV release. Preventing the disposal of the pro-apoptotic factors in combination with BCL-2 inhibition sensitizes AML FLT3-ITD cells to apoptosis. Additional evaluation in primary patient material and further studies to identify the factors responsible for sensitization to apoptosis upon EV release inhibition are ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of the Menin-MLL1 Interaction in MLL-Rearranged Primary Mixed-Phenotype Acute Leukemia Samples Promotes Leukemic Differentiation

Mixed phenotype acute leukemias (MPALs) simultaneously present features typical for more than one hematological cell lineage. The rarity of such patients combined with the lack of well-defined diagnostic criteria have so far prevented the design of optimal therapy regimens resulting in the occurrence of lineage switch and therewith associated treatment failures. Rearrangements involving MLL gene (also known as KMT2A) occur in around 15% of MPAL patients (typically infants) and are associated with a very poor outcome. Moreover, several studies demonstrated dependency of MLL-rearranged (MLL-r) leukaemias on the interaction between N-terminus of MLL with Menin and its successful therapeutic targeting by small molecular weight compounds, that are currently validated in clinical trials. Here we investigated potential application of the Menin-MLL inhibitor SNDX-5613 in MLL/AF4 MPAL infants who presented with the bilineage B/Myeloid phenotype. Using a co-culture approach with human bone marrow-derived mesenchymal stromal cells (MSCs) and media optimized for growth of lymphoid and myeloid lineages, we were able to expand and monitor primary MLL-r patient material for more than three weeks. Primary material from two MPAL patients and their lymphoid and myeloid subpopulations were analyzed for their drug responses including immunophenotypic and global transcriptomic characterization. We observed a dramatic loss of cell numbers in response to SNDX-5613, with the IC50 value ranging from 80 to 800nM. Similar analyses of the sorted fractions revealed that the observed loss of cell numbers is mainly caused by the reduction of the lymphoid subfraction. Immunophenotypic analyses showed that surviving cells within both lymphoid and myeloid fractions are losing CD34 with simultaneous gain of the CD38 marker, suggesting cellular maturation. Treatment of the lymphoid fraction with SNDX-5613 caused a dose-dependent loss of the CD19+CD33+ cell population and a simultaneous gain of CD19+CD33-, and CD19-CD33+ cells. These data suggest the cellular origin of the myeloid fraction to be residing within the lymphoid counterpart. Interestingly, myeloid cells responded to Menin-MLL inhibition with increased surface expression of monocytic markers (e.g. CD11b, CD14, CD15), elevated RNA expression of genes involved in cell adhesion and morphological changes, indicating myeloid differentiation of the surviving cells. Moreover, transcriptomic analysis of the lymphoid subfraction revealed dose-dependent increase of the MS4A1 gene expression (encoding for CD20) suggesting simultaneous lymphoid maturation of the CD19+ proB-like blasts. In conclusion, our study shows that Menin-MLL inhibition drives both myeloid differentiation and lymphoid maturation in MLL-r MPALs. These myeloid data are consistent with observations of differentiation syndrome as an on-target effect in current clinical trials while the phenotypic changes in the lymphoid cells are new findings. Together, these data strongly suggests that MLL-r MPAL patients could benefit from the inclusion of the Menin-MLL1 inhibitors into their treatment regimens.

Abstract 2059: Immunotherapy testing in 3D Ex vivo Patient Tissue Platform with preserved tumor microenvironment

Abstract Introduction Broad application of immunotherapy to treat cancer has been hampered by low response rates in the clinics (~20%) and high failure rates of clinical trials. The lack of translational preclinical models that accurately replicate human immunological complexity is one of the reasons leading to ineffective candidates in clinical trials. There is an urgent need of advanced preclinical models that can enhance clinical trial success rate and improve clinical impact of immunotherapies. We present a novel 3D Ex vivo Patient Tissue platform combining short-term 3D ex vivo tumor culture system with high content image (HCI)-based analysis. The platform preserves tumor heterogeneity and tumor immune context, including (exhausted) tumor infiltrating lymphocytes. We report a quantification of ex vivo tumor responses to a panel of immunotherapies, including checkpoint inhibitors, tested on patient tissues from various cancers. Methods Patient tumor tissues were obtained from hospitals and tissue providers and processed within 24 hours to preserve the tumor microenvironment (TME). Tumor clusters from ovarian, cervical, breast, and non-small cell lung cancer patients were embedded in a protein-rich hydrogel and exposed to panels of immunomodulatory drugs in a 384-well format for 5-7 days. Phenotypic effects of the drugs on physiologically relevant morphological changes, such as tumor cell killing and immune cell proliferation, were measured using our proprietary automated HCI analysis platform. In addition, IHC and FACS analysis of primary samples as well as cytokine measurements were performed. Results Responses of ex vivo tissues to immunotherapies targeting various pathways (e.g., ipilimumab, pembrolizumab and STING agonists), combination treatments and selected controls were evaluated by automated phenotypic analysis platform. Based on their sensitivity, patient tissues were classified as immunotherapy responders or non-responders. A deeper investigation of the responder tissues was performed with IHC and FACS to pinpoint the critical TME components necessary for immunotherapy sensitivity. In addition, cytokine profiling was done in supernatants from treated ex vivo cultures to confirm the functional HCI readouts. Accurate and reproducible response evaluation demonstrated the feasibility of preclinical immunotherapy drug testing on primary patient material using this platform. Conclusion The 3D ex vivo patient tissue platform successfully combined drug testing protocols using fresh patient tumor tissue with preserved TME and advanced 3D HCI analysis. The platform offers a rapid, reliable and patient-relevant approach to test (clinical) immunotherapies for different solid tumours. It has the potential to significantly improve the preclinical evaluation of immunotherapies and support the decision-making process during progression of drug candidates to the clinic. Citation Format: Nataliia Beztsinna, Niels Meesters, Lidia Daszkiewicz, Fanny Grillet, Donny van der Meer, Kuan Yan, Emma Spanjaard, Willemijn Vader, Leo Price. Immunotherapy testing in 3D Ex vivo Patient Tissue Platform with preserved tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2059.

Stem Cell Based Models in Congenital Hyperinsulinism - Perspective on Practicalities and Possibilities.

Congenital hyperinsulinism (CHI) is a severe inherited neonatal disorder characterized by inappropriate insulin secretion caused by genetic defects of the pancreatic beta cells. Several open questions remain in CHI research, such as the optimal treatment for the most common type of CHI, caused by mutations in the genes encoding ATP-sensitive potassium channels, and the molecular mechanisms of newly identified CHI genes. Answering these questions requires robust preclinical models, particularly since primary patient material is extremely scarce and accurate animal models are not available. In this short review, we explain why pluripotent stem cell derived islets present an attractive solution to these issues and outline the current progress in stem-cell based modeling of CHI. Stem cell derived islets enable the study of molecular mechanisms of CHI and the discovery of novel antihypoglycemic drugs, while also providing a valuable model to study the biology of variable functional states of beta cells.

Blast cells surviving acute myeloid leukemia induction therapy are in cycle with a signature of FOXM1 activity

BackgroundDisease relapse remains common following treatment of acute myeloid leukemia (AML) and is due to chemoresistance of leukemia cells with disease repopulating potential. To date, attempts to define the characteristics of in vivo resistant blasts have focused on comparisons between leukemic cells at presentation and relapse. However, further treatment responses are often seen following relapse, suggesting that most blasts remain chemosensitive. We sought to characterise in vivo chemoresistant blasts by studying the transcriptional and genetic features of blasts from before and shortly after induction chemotherapy using paired samples from six patients with primary refractory AML.MethodsLeukemic blasts were isolated by fluorescence-activated cell sorting. Fluorescence in situ hybridization (FISH), targeted genetic sequencing and detailed immunophenotypic analysis were used to confirm that sorted cells were leukemic. Sorted blasts were subjected to RNA sequencing. Lentiviral vectors expressing short hairpin RNAs were used to assess the effect of FOXM1 knockdown on colony forming capacity, proliferative capacity and apoptosis in cell lines, primary AML cells and CD34+ cells from healthy donors.ResultsMolecular genetic analysis revealed early clonal selection occurring after induction chemotherapy. Immunophenotypic characterisation found leukemia-associated immunophenotypes in all cases that persisted following treatment. Despite the genetic heterogeneity of the leukemias studied, transcriptional analysis found concerted changes in gene expression in resistant blasts. Remarkably, the gene expression signature suggested that post-chemotherapy blasts were more proliferative than those at presentation. Resistant blasts also appeared less differentiated and expressed leukemia stem cell (LSC) maintenance genes. However, the proportion of immunophenotypically defined LSCs appeared to decrease following treatment, with implications for the targeting of these cells on the basis of cell surface antigen expression. The refractory gene signature was highly enriched with targets of the transcription factor FOXM1. shRNA knockdown experiments demonstrated that the viability of primary AML cells, but not normal CD34+ cells, depended on FOXM1 expression.ConclusionsWe found that chemorefractory blasts from leukemias with varied genetic backgrounds expressed a common transcriptional program. In contrast to the notion that LSC quiescence confers resistance to chemotherapy we find that refractory blasts are both actively proliferating and enriched with LSC maintenance genes. Using primary patient material from a relevant clinical context we also provide further support for the role of FOXM1 in chemotherapy resistance, proliferation and stem cell function in AML.

Repeat to gene expression ratios in leukemic blast cells can stratify risk prediction in acute myeloid leukemia

BackgroundRepeat elements constitute a large proportion of the human genome and recent evidence indicates that repeat element expression has functional roles in both physiological and pathological states. Specifically for cancer, transcription of endogenous retrotransposons is often suppressed to attenuate an anti-tumor immune response, whereas aberrant expression of heterochromatin-derived satellite RNA has been identified as a tumor driver. These insights demonstrate separate functions for the dysregulation of distinct repeat subclasses in either the attenuation or progression of human solid tumors. For hematopoietic malignancies, such as Acute Myeloid Leukemia (AML), only very few studies on the expression/dysregulation of repeat elements were done.MethodsTo study the expression of repeat elements in AML, we performed total-RNA sequencing of healthy CD34 + cells and of leukemic blast cells from primary AML patient material. We also developed an integrative bioinformatic approach that can quantify the expression of repeat transcripts from all repeat subclasses (SINE/ALU, LINE, ERV and satellites) in relation to the expression of gene and other non-repeat transcripts (i.e. R/G ratio). This novel approach can be used as an instructive signature for repeat element expression and has been extended to the analysis of poly(A)-RNA sequencing datasets from Blueprint and TCGA consortia that together comprise 120 AML patient samples.ResultsWe identified that repeat element expression is generally down-regulated during hematopoietic differentiation and that relative changes in repeat to gene expression can stratify risk prediction of AML patients and correlate with overall survival probabilities. A high R/G ratio identifies AML patient subgroups with a favorable prognosis, whereas a low R/G ratio is prevalent in AML patient subgroups with a poor prognosis.ConclusionsWe developed an integrative bioinformatic approach that defines a general model for the analysis of repeat element dysregulation in physiological and pathological development. We find that changes in repeat to gene expression (i.e. R/G ratios) correlate with hematopoietic differentiation and can sub-stratify AML patients into low-risk and high-risk subgroups. Thus, the definition of a R/G ratio can serve as a valuable biomarker for AML and could also provide insights into differential patient response to epigenetic drug treatment.

Relapsing Pediatric T-Cell Acute Lymphoblastic Leukemia Downregulates T-Cell Properties and Upregulates Cell Adhesion

Relapse is the main cause of death from pediatric acute precursor T-cell leukemia (T-ALL), but the mechanisms underlying relapse remain poorly understood. We performed an integrated longitudinal multi-level omics analysis of 12 T-ALL patients to reveal common relapse-driving processes. Using high-throughput NGS-based techniques we compared the mutation (SNV/InDels) and copy number alteration (CNA) patterns as well as gene expression and methylation levels at initial diagnosis, remission and relapse in primary patient material and in patient derived xenografts (PDX).The PDX models largely recapitulated the clonal architecture of the primary leukemias. Aberrant expression of T-ALL transcription factors (TAL1, TAL2, LY1L, LMO2, TLX1, TLX 3, NKX2.4, NKX2.5) was preserved from initial presentation to relapse in all of the patients, as were most of the large CNA and frequent leukemia-specific SNVs and InDels. The known activating mutations in NT5C2 were acquired at relapse by 7/12 patients. RNA-seq analysis showed 40 transcripts to be either down- or upregulated at the time of relapse in ≥4 patients. Methylome analysis of approximately 25,700 promoters showed 141 (0.6%) to be significantly altered (∆ ß ≥0.2; McNemar0.05), of which 109 were recurrently hypomethylated and 32 hypermethylated at relapse.Functional enrichment classification revealed that 12 of the 32 promoters that were hypermethylated in relapse were highly T-cell specific and regulate genes involved in lymphopoiesis (DNTT, CD3G, CD3D, PTCRA, RAG2, RHOH, SIT1), encoding T cell receptors (TRBV30, TRBJ2-1, TRBV2, TRBV28) and the T-Cell Leukemia Homeobox 3 gene. Similarly, ontology terms associated with T-cell signaling and hematopoiesis were overrepresented among genes whose RNA expression was downregulated at relapse in ≥3 patients (CD4, CD8, CD9, DNTT, IL4, PTCRA RAG1, RAG2, TRAV13-2, TRAV18 and THEMIS). These changes in RNA expression and promoter methylation were reflected by cell surface marker expression in primary patient derived cells obtained at the time of initial presentation and relapse.When considering the genes with significantly relapse-specific hypomethylated promoters and overexpressed transcripts, we identified genes associated with homophilic cell adhesion via plasma membrane adhesion molecules (GO:0007156) and containing cadherin conserved site (enrichment score for expression: 4.98 and for hypomethylation 2.39). Moreover, three of the patients acquired at relapse missense mutations in several cadherin-like genes (CDH23, CELSR1, PCDHA1, PCDHA10, PCDHA6, PCDHAC1, PCDHAC2 ; enrichment score: 2.47), although further studies are required to determine the functional consequences of these SNVs. Interestingly, the Suppressor Of Cytokine Signaling 2 (SOCS2) mRNA was the most frequently recurrent overexpressed transcript at relapse in 5/12 patients. SOCS2 is a negative regulator of cytokine receptor signaling via the JAK/STAT pathway and its deregulation was previously identified as part of a stemness-related molecular signature signifying unfavorable outcome in acute myeloid and lymphoblastic leukemia (Vitali et. al, 2015).In conclusion, the longitudinal analysis of this cohort of patients identified relapse-specific alterations with potential functional impact. We postulate that relapsed T-ALLs lose T-cell specific properties and gain properties that result in resistance against chemotherapy. The latter may be related to the ability of relapse cells to upregulate the expression of cell adhesion molecules such as cadherins, protocadherins and integrins thus putatively modulating the leukemia/stroma interaction. DisclosuresSchrappe:Baxalta: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; Medac: Consultancy, Research Funding.

Novel Mutations in RASGRP1 Are Associated with Immune Dysregulation and Predispose to EBV-Induced Lymphoma

& I. Somekh and B. Marquardt contributed equally§ C. Klein and R. Somech contributed equallyBackgroundRAS guanyl-releasing protein 1 (RASGRP1) deficiency has recently been shown to cause a primary immunodeficiency disorder in one patient (Salzer et al, Nat Immunol 2016). Clinically, the disease was characterized by recurrent episodes of pneumonia, bronchiectasis, severe failure to thrive, CD4 T-cell lymphopenia, elevated TCRγδ cell proportions as well as low-grade Epstein-Barr virus (EBV)-associated B cell lymphoma. Immunologically, RASGRP1 deficiency is associated with defective proliferation and activation of T and B cells, respectively. Here, we report 3 novel patients with RASGRP1 deficiency from 2 unrelated consanguineous kindred.MethodsOne patient of Turkish origin (P1) and 2 Palestinian 1st degree cousins (P2, P3) were evaluated. Genetic analysis using whole exome sequencing was conducted. Immunological and biochemical assays were performed on primary patient material and genetically engineered T-cell lines using CRISPR/Cas9.ResultsClinical findings for all three patients included failure to thrive, hepatosplenomegaly, lymphadenopathy, and recurrent pulmonary infections resulting in bronchiectasia. Two patients had overt auto-immunity (AIHA and autoimmune thrombocytopenia, respectively). One patient had increased anti-nuclear antibody titers. One patient (P1) developed EBV-associated diffuse large B cell lymphoma. He underwent autologous hematopoietic stem cell transplant due to relapse after combined rituximab and NHL-BFM-90 treatment protocol and is currently in remission at the age of 14 years. P2 had deceased at the age of 3 years due to disseminated intravascular coagulation. P3 developed diffuse large B cell lymphoma at the age of 3 years, treated with R-CHOP protocol and is currently in remission.Immune workup of P2 and P3 revealed low levels of T-cell receptor excision circles (TREC), an abnormal T-cell receptor (TCR) Vß repertoire and a markedly increased proportion of TCRγδ cells. Phytohemagglutinin (PHA)-induced lymphocyte proliferation and anti-CD3 mitogen proliferation responses were decreased. EBV-viral load in peripheral blood was elevated for both patients, as measured by PCR.Genetic analysis by whole exome sequencing identified an autosomal recessive homozygous mutations in RASGRP1. P1 had an inversion (c.649_650inv; p.Glu217Ser) and P2/P3 had a deleterious frameshift mutation (c.1111_1114del; p.Asp371Ilefs*7). The variants were confirmed by Sanger sequencing and segregated with the disease phenotype. To confirm the functional relevance of the RASGRP1 mutations, we engineered Jurkat cells via CRISPR/Cas9 editing. RASGRP1 -/- Jurkat cells showed decreased phosphorylation of ERK1/2 and decreased CD69 expression in comparison to wildtype cells. Jurkat cells with RASGRP1 knockin mutants are currently under analysis.ConclusionsRASGRP1 deficiency is a novel primary immunodeficiency disorder associated with immune dysregulation and susceptibility to EBV-induced B-cell malignancies. Early diagnosis of RASGRP1 deficiency is critical. Even though clinical remission of EBV-lymphoma may be reached using high dose chemotherapy regimens, long term cures may only be reached by allogeneic hematopoietic stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic Efficacy of Mcl-1 Antagonism in Large Granular Lymphocyte Leukemia

Introduction: Large granular lymphocyte (LGL) leukemia is a spectrum of rare clonal lymphoproliferative disorders, all of which involve expansion of large granular lymphocytes. Immunosuppressive regimens using methotrexate or cyclophosphamide are utilized as front-line treatments in LGL leukemia but are not curative nor are they effective in all patients. The aggressive LGL variants have no effective treatment and are refractory to conventional chemotherapy. The lack of effective and targeted therapies results from an incomplete picture of survival mechanisms that contribute to the pathogenesis of leukemic LGLs.We have previously reported that STAT3 is constitutively active in LGL leukemia cells and up to 40% of patients harbor activating mutations in STAT3 within the leukemic clone. We have also reported that the pro-survival Bcl-2-family protein Mcl-1 is upregulated in LGL leukemia patients. Additionally, we have identified MCL1 as a transcriptional target of STAT3 in leukemic LGLs. Recently, novel small molecule inhibitors of Mcl-1 have been identified including marinopyrrole A (maritoclax) and its derivative KS18. These inhibitors effectively induced apoptosis in Mcl-1-dependent cells and suppressed the growth of Mcl-1-dependent hematologic cancer xenografts in nude mice without apparent toxicity. Thus, this study further investigates the role of Mcl-1 in LGL leukemia and the potential therapeutic application of the Mcl-1 antagonists maritoclax and KS18.Results: Knockdown of Mcl-1 in human-derived LGL leukemia cell lines (TL1 and NKL) resulted in decreased cell viability by MTS assay and increased apoptosis by flow cytometry in Mcl-1 siRNA versus scramble siRNA-transfected cells (NKL and TL-1 cells: p < 0.001). Treatment with maritoclax or KS18 decreased cell proliferation and increased apoptosis in NKL, TL1 and RNK16 (rat-derived LGL leukemia cell line) cells (Maritoclax, EC50: 2.45, 1.43 and 1.23 μM respectively) (KS18, EC50: 1.30, 0.60 and 0.31 μM respectively). Ex vivo treatment with maritoclax and KS18 induced selective apoptosis in PBMCs from LGL leukemia patients (NK-LGL, n = 6; T-LGL, n = 12) but not normal donor PBMCs (n = 4) (Maritoclax and KS18, p < 0.0001). Importantly, there was no significant difference in treatment efficacy in patient cells with wild-type STAT3 (n = 9) versus mutant STAT3 (Y640F, n = 5; D661Y, n = 3). We then proceeded to mechanistic studies of Mcl-1 antagonism. Both maritoclax and KS18 induced selective and dose-dependent Mcl-1 degradation by western blot in NKL, TL1, RNK16 and PBMCs from LGL leukemia patients. We further delineated that this effect was proteasome dependent as it is reversible by pre-treatment with MG132, a proteasome inhibitor.Preclinical in vivo studies were then conducted in Fisher F344 rats that were transplanted with primary leukemic LGL cells and monitored for signs of leukemia through serial CBC and body weight measurements. Leukemic rats developed progressive anemia, thrombocytopenia and leukocytosis and significant weight loss. Rats were treated with vehicle or KS18 (2.5 or 5.0 mg/kg i.p. daily) upon detection of leukemic features. In non-leukemic KS18-treated rats, there were no observed toxicities and there was no difference in survival or weight gain versus vehicle-treated non-leukemic rats. However, leukemic KS18-treated rats exhibited findings consistent with tumor lysis syndrome, including sudden death associated with severely increased serum LDH and increased apoptosis in heart and spleen tissues. At necropsy, leuk/KS18 rats exhibited significantly decreased infiltration and elevated apoptosis of leukemic cells in heart and spleen tissues. Conversely, in leuk/veh rats, spleen tissue lacked normal histologic structure with red pulp replaced almost completely by leukemia cells. Ex vivo KS18 treatment also decreased the viability of PBMC and spleen cells from leukemic animals (p < 0.0001). These data indicate that Mcl-1 is a promising therapeutic target as evidenced by potent therapeutic efficacy of KS18 in LGL cell lines and primary patient material and in an aggressive rat model of LGL leukemia. Further studies are required in order to optimize drug dose, timing and concentrations of KS18 in order to prevent potential tumor lysis syndrome and optimize therapeutic benefit. DisclosuresGru:Seattle Genetics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees.

C-Terminal BRE Overexpression in 11q23-Rearranged and t(8;16) Acute Myeloid Leukemia Is Caused By Intragenic Transcription Initiation

Abstract TextOverexpression of the BRE (Brain and Reproductive organ-Expressed ) gene defines a distinct pediatric and adult acute myeloid leukemia (AML) subgroup (Balgobind et al, Leukemia 2010, and Noordermeer et al, Blood 2011 and 2012). BRE overexpression specifically associates with 11q23/MLL/KMT2A-rearranged AML, occurring in 70% and 50% of 11q23 childhood and adult AML cases, respectively. In adult 11q23-rearranged AML, BRE overexpression significantly associates with younger age. BRE functions in BRCA1-A (Breast Cancer 1) complex mediated DNA damage repair, BRISC (BRCC3 isopeptidase complex) complex mediated mitotic spindle assembly and in protection from apoptosis and senescence. Another constituent of the BRCA1-A/BRISC complexes is BRCC3 which is recurrently mutated in AML, especially in cases with a TET2 mutation (Huang et al., Haematologica, 2015). BRE is relatively highly expressed in blood cells in general but its exact function in hematopoiesis is unknown.By performing genome-wide ChIP-sequencing analyses on primary KMT2A-MLLT3 patient material (n=9) we identified a promoter enriched for active chromatin marks (H3K4me3/H3K27Ac) in BRE intron 4, exclusively in AML cases with BRE overexpression. Both 5' RACE end RNA-sequencing identified strong biallelic expression of a previously unknown C-terminal BRE transcript. This transcript starts near the intragenic promoter, with BRE intron 4 sequences spliced to exon 5 and downstream sequences. RNA-seq and qRT-PCR showed that the downstream transcript is expressed 5-8-fold higher compared to the full-length transcript, explaining BRE overexpression. Of note, two start codons in exon 5 of the downstream BRE transcript may cause the formation of N-terminally truncated BRE isoforms.By performing a qRT-PCR that specifically recognizes the new BRE transcript on samples from a large cohort of pediatric and adult AML (n=195) we observed that it was highly expressed in 60% of 11q23/ KMT2A -rearranged (n=58) and 70% of t(8;16)/ KAT6A-CREBBP cases (n=20), while it was virtually absent from other AML subsets (n=101) and normal hematopoietic tissues (n=14). In gene reporter assays, the AML-specific fusion protein KMT2A-MLLT3 transactivated the intragenic BRE promoter. Further epigenome analyses revealed 97 additional intragenic promoter marks frequently bound by KMT2A in AML with C-terminal BRE overexpression. The bound genes may be part of a context-dependent KMT2A-MLLT3-driven oncogenic program, because they were higher expressed in this AML subtype compared to other cytogenetically-defined AML groups (p<0.01). C-terminal BRE might be a contributor to this oncogenic program because in a case with relapsed AML, we observed an ins(11;2) fusing CHORD1 in frame to BRE exon 5. The CHORDC1 fusion to BRE exon 5 is remarkable because the alternate BRE transcript starts with exon 1b also fused to exon 5. This means that if the CHORDC1-BRE and alternate BRE transcripts would be translated, they would contain a similar C-terminal part of the BRE protein. The CHORDC1 disruption may also contribute to AML pathogenesis because it was recently shown that heterozygous Chord1 (a.k.a. Morgana) inactivation in mice results in the spontaneous development of atypical chronic myeloid leukemia (Di Savino, Blood, 2015). We conclude that abnormal expression of C-terminal alternate BRE sequences specific for AML is caused by distinct, non-random events; by a chromosomal abnormality and by intragenic transcription initiation in 11q23/ KMT2A -rearranged and t(8;16)/ KAT6A-CREBBP AML. These findings warrant further studies to determine whether leukemia-specific alternate BRE contributes to disease pathogenesis and how abnormal intragenic transcription activation in AML is brought about. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53

Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein–protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.

Establishment of patient-derived cancer organoids for drug-screening applications.

Adult stem cell-based organoid technology is a versatile tool for the generation and long-term maintenance of near-native 3D epithelial tissues in vitro. The generation of cancer organoids from primary patient material enables a range of therapeutic agents to be tested in the resulting organoid cultures. Patient-derived cancer organoids therefore hold great promise for personalized medicine. Here, we provide an overview of the protocols used by different groups to establish organoids from various epithelial tissues and cancers, plus the different protocols subsequently used to test the in vitro therapy sensitivity of these patient-derived organoids. We also provide an in-depth protocol for the generation of head and neck squamous cell carcinoma organoids and their subsequent use in semi-automated therapy screens. Establishment of organoids and subsequent screening can be performed within 3 months, although this timeline is highly dependent on a.o. starting material and the number of therapies tested. The protocol provided may serve as a reference to successfully establish organoids from other cancer types and perform drug screenings thereof.

Abstract 3250: CD229-targeted CAR T cell therapy for the treatment of B cell lymphoma

Abstract Background: B cell lymphomas are the most common hematologic malignancy. Unfortunately, therapy-resistant relapses, even after CD19 CAR T cells, drive poor survival in certain lymphoma subtypes. CD229 is an immune receptor that is expressed on normal B cells as well as various B cell malignancies. In this study, we performed the first preclinical evaluation of CD229 as a therapeutic CAR T cell target in B cell lymphoma. Methods: We analyzed expression of CD229 in B cell lymphoma cell lines and primary patient material by semi-quantitative PCR, western blot and flow cytometry. We then generated CD229 CAR T cells using our own, previously described CD229-specific scFv (single-chain fragment variable) antibody and CD3ζ / 4-1BB signaling domains. We determined the efficacy of CD229 CAR T cells against B cell lymphoma cell lines and primary patient material in vitro using established cytotoxicity assays and cytometric bead arrays. Finally, we utilized B cell lymphoma cell line-derived xenograft mouse models to determine the in vivo efficacy of CD229 CAR T cells. Results: Expression analysis of 12 B cell lymphoma cell lines including diffuse large B cell lymphoma (DLBCL), Burkitt lymphoma, B cell acute lymphoblastic leukemia (B-ALL) and mantle cell lymphoma revealed consistent expression of CD229 transcript, total protein and surface protein. Similarly, expression analysis of primary material from patients with B cell lymphoma revealed CD229 surface expression in 16/16 cases of B cell chronic lymphoblastic leukemia (B-CLL), 13/13 mantle cell lymphomas, 6/6 follicular lymphomas and 4/4 DLBCLs. Next, we efficiently generated CAR T cells targeting CD229 and utilized these CAR T cells for in vitro cytotoxicity assays. Coculture of CD229 CAR T cells with B cell lymphoma cell lines resulted in effective eradication of B cell lymphoma cells (two DLBCL, two mantle cell lymphoma and two Burkitt lymphoma) at low effector-target ratios while control CAR T cells had no effect. In addition to robust killing of B cell lymphoma cell lines, CD229 CAR T cells were also able to effectively kill primary cells from 4/4 mantle cell lymphoma patients and 3/3 B-CLL patients. CD229 CAR T cells produced high amounts of proinflammatory type-1 cytokines including IFNγ, IL2, and TNFα upon exposure to B cell lymphoma cell lines and primary material. Finally, we observed efficient tumor control by CD229 CAR T cells, but not control CAR T cells, using multiple B cell lymphoma xenograft mouse models. Conclusions: Taken together, we demonstrate for the first time that CD229 CAR T cells are a promising candidate for the treatment of B cell lymphoma as evidenced by the consistent expression of CD229 on B cell lymphoma cells and the efficacy of CD229 CAR T cells against B cell lymphoma in vitro and in vivo. Citation Format: Michael L. Olson, Sabarinath V. Radhakrishnan, Tim C. Luetkens, Djordje Atanackovic. CD229-targeted CAR T cell therapy for the treatment of B cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3250.

Abstract 4232: More than a bridging therapy: Targeting CD123 with rapidly switchable universal CAR-T cells for treatment of acute leukemia

Abstract Chimeric antigen receptor T-cell (CAR-T) therapy targeting CD19 or BCMA is tremendously effective in treating hematological malignancies. However, the application of CAR-T beyond certain B-cell malignancies such as ALL, MM, lymphoma and CLL remains challenging due to the lack of tumor-specific antigens and the limited ability to control acute and potential long term CAR-T reactivity in patients. Therefore, a rapidly switchable universal CAR-T platform (UniCAR) was developed. In this system, antigen-specificity is provided by soluble adaptors of short pharmacokinetic half-life, termed targeting modules (TM), which redirect T-cells engineered to express a universal CAR against tumors, allowing for excellent controllability of CAR-T reactivity while maintaining the high anti-tumor activity of CAR-T cells. Here we present results from late stage pre-clinical characterization of UniCAR-T targeting CD123 (UniCAR-T-CD123) for treatment of acute leukemia. Pre-clinical characterization confirmed specificity and safety of the approach. UniCAR-T proofed to be per se inert and safe in clinically relevant in vivo toxicity models. Activation of UniCAR-T occurs solely in the presence of the CD123-specific TM (TM123) and upon cross-linkage to CD123 expressing leukemic cells. Specific lysis of leukemic target cell lines and primary patient material is induced in a dose-dependent manner at pico-molar TM123 concentrations. Notably, induction of cytokine release occurs at higher TM doses than on-set of target cell lysis, opening a therapeutic window for clinical application. In vivo efficacy of UniCAR-T re-directed against CD123 was proven in CDX and PDX mouse models. Interestingly, anti-leukemic responses of switch-controllable UniCAR-T were demonstrated to be comparable to conventional CD123-specific CAR-T in vitro at comparable doses. In contrast to conventional CD123 CAR-T, CD123-specific toxicity of TM-activated UniCAR-T towards hematopoietic progenitors was reversible and could be abrogated by withdrawal of TM, allowing for normal development of human hematopoiesis in a xenograft model. In summary, in vitro and in vivo evidence suggests that UniCAR-T-CD123 could provide strong efficacy against CD123 expressing hematological malignancies while providing excellent control and ensuring recovery of normal hematopoiesis post treatment. A phase IA dose-finding study is ongoing. Citation Format: Simon Loff, Jan-Erik Meyer, Johannes Spehr, Julia Riewaldt, Cordula Gründer, Maria Schreiber, Michael Bachmann, Michael Pehl, Gerhard Ehninger, Armin Ehninger, Marc Cartellieri. More than a bridging therapy: Targeting CD123 with rapidly switchable universal CAR-T cells for treatment of acute leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4232.

Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids.

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the increasing use of organoids as a culturing method for primary patient material in the last years, efficient transfer methods of components for genetic engineering in this 3D culture system are in need. Especially for organoids, the efficiency of genetic manipulations depends on a successful transfection. Thus, this protocol was developed to facilitate the electroporation of organoids and to prove its universal functionality in different entities. Human colorectal, pancreatic, hepatic and gastric cancer organoids were successfully electroporated with small and large plasmids in comparison. Based on GFP encoding vectors, the transfection efficiency was determined by FACS. No extensive preparation of the cells or special, cost-intensive electroporation buffers are necessary, and the protocol can be performed within one day.