Anti-leukemia Immune Response Research Articles

Clinical efficacy and immune response of BCL-2 inhibitors combined with hypomethylating agents in the treatment of acute myeloid leukemia

ObjectiveAcute myeloid leukemia (AML) is a malignant clonal proliferative disease with a high mortality rate. The combination therapy of BCL-2 inhibitor Venetoclax (VEN) and hypomethylating agents (HMAs) has significant anti-leukemia activity.MethodsWe analyzed the efficacy, safety and immune response characteristics of AML patients who were unfit for high-dose chemotherapy and accepted the medication of VEN + HMAs.ResultsAfter VEN + HMAs treatment, 31 newly diagnosed AML patients had the morphologic leukemia-free state rate (MLFS%) of 80.6% (25/31), complete response rate (CR%) of 54.8% (17/31), the minimal residual disease negative rate (MRD-%) of 51.6% (16/31), and the median progression-free survival (PFS) of 14 months. After treatment, the proportion of bone marrow primitive cells, the MRD level, white blood cell (WBC) count, fibrinogen (FIB) level and the proportion of B cells were significantly decreased. The red blood cell (RBC) count, hemoglobin (HGB) level, platelet count (PLT) count, activated partial thromboplastin time (APTT), the proportion of total T cells, CD8 + T cells and the IFN-γ level were significantly increased. After VEN + HMAs treatment, 12 relapsed AML patients had a MLFS% of 50% (6/12), CR% of 33.3% (4/12), MRD-% of 25% (3/12), and a median PFS of 7 months. After treatment, the proportion of bone marrow primitive cells and MRD level were slightly decreased, the proportions of CD8 + T cells and NK cells were significantly increased, the proportion of B cells and IL-10 level were significantly decreased. 12 AML patients who receive microtransplantation (MST) treatment using VEN + HMAs as a pretreatment regimen had a PFS of 20.5 months, which was much greater than VEN + HMAs group alone. Hematological recovery was better in the MST group with significantly increased RBC count, HGB level and PLT count. The most common adverse events were myelosuppression, agranulocytosis, infection and cardiovascular toxicity. No fatal adverse events were reported.ConclusionThe combination of BCL-2 inhibitors and HMAs had good efficacy and safety in AML patients who were unfit for high-dose chemotherapy, which may improve the immune microenvironment and enhance anti-leukemia immune response.

IVAC-XS15-CLL01: personalized multi-peptide vaccination in combination with the TLR1/2 ligand XS15 in CLL patients undergoing BTK-inhibitor-based regimens.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia among adults in Western countries. Despite the introduction of targeted therapies, including first-line Bruton's tyrosine kinase inhibitor (BTKi) treatment, CLL remains largely incurable. Frequent disease relapses occur due to remaining treatment-resistant CLL cells, calling for novel therapies to eliminate minimal residual disease (MRD). Peptide-based vaccination targeting human leucocyte antigen (HLA)-presented CLL-associated antigens represents a promising, low-side-effect therapeutic option to optimize treatment responses and eliminate residual tumor cells by inducing an anti-leukemic immune response. The iVAC-XS15-CLL01 trial is an open-label, first-in-human (FIH) Phase I trial, evaluating the CLL-VAC-XS15 vaccine in CLL patients undergoing BTKi-based therapy. The vaccine was developed from HLA-presented CLL-associated antigen peptides, identified through comparative mass-spectrometry-based immunopeptidome analyses of CLL versus healthy samples in a previous study. To facilitate rapid and cost-effective deployment, vaccine peptides are selected for each patient from a pre-manufactured "peptide warehouse" based on the patient's individual HLA allotype and CLL immunopeptidome. The trial enrolls 20 CLL patients, who receive up to three doses of the vaccine, adjuvanted with the toll-like-receptor (TLR) 1/2 ligand XS15 and emulsified in Montanide ISA 51 VG. The primary objective of the iVAC-XS15-CLL01 trial is to assess the safety and immunogenicity of the CLL-VAC-XS15 vaccine. Secondary objectives are to evaluate the vaccine impact on MRD, progression-free survival, and overall survival, as well as comprehensive immunophenotyping to characterize vaccine-induced T-cell responses. This Phase I trial aims to advance CLL treatment by enhancing immune-mediated disease clearance and guiding the design of subsequent Phase II/III trials to implement a new therapeutic strategy for CLL patients.

Recent Advances in Immune-Based Therapies for Acute Myeloid Leukemia.

Despite advancements, acute myeloid leukemia (AML) remains unconquered by current therapies. Evidence of immune evasion during AML progression, such as HLA loss and T-cell exhaustion, suggests that antileukemic immune responses contribute to disease control and could be harnessed by immunotherapy. In this review, we discuss a spectrum of AML immunotherapy targets, encompassing cancer cell-intrinsic and surface antigens as well as targeting in the leukemic milieu, and how they can be tailored for personalized approaches. These targets are overviewed across major immunotherapy modalities applied to AML: immune checkpoint inhibitors, antibody-drug conjugates, therapeutic vaccines, bispecific/trispecific antibodies, and chimeric antigen receptor (CAR)-T and CAR-NK cells. Significance: Immune therapies in AML treatment show evolving promise. Ongoing research aims to customize approaches for varied patient profiles and clinical scenarios. This review covers immune surveillance mechanisms, therapy options like checkpoint inhibitors, antibodies, CAR-T/NK cells, and vaccines, as well as resistance mechanisms and microenvironment considerations.

Abstract 5377: Investigating human leukocyte antigen (HLA) class 1 leukemogenic pathways in acute myelogenous leukemia

Abstract Background: Aging, smoking and germline predisposition are known to accelerate leukemogenesis. However, the the ability for myeloid clonal pathogenic variants (MCPV) to elicit an antigen-driven host antileukemia immune response is not well characterized. A better understanding on how cellular immunity selectively discriminates leukemia-related “neopeptides” could uncover mechanisms supporting tumor genome evolution. Methods: 122 patients with acute myelogenous leukemia (AML) were included and 111 had available protein coding sequences. A neopeptide library was created using protein FASTA coding extracted for individual myeloid clonal mutations via National Center for Biotechnology Information (NCBI). Patient class 1 HLA typing was obtained through Tempus, and Genomic Testing Cooperative laboratories. Prediction of number and strength of AML neopeptide bindings to MHC class I molecules were obtained via NetMHCPan-1. Analysis was performed with K-means algorithm assigning age at diagnosis, detected MCPV, number of neopeptides binding (as continuous variable), overall survival (OS) and event-free survival (EFS) as variables. Unsupervised data input allowed 3 cluster partitions for differential survivals allowing HLA class 1 peptide binding aggregation with potential roles in leukemogenesis and patient outcome. Descriptive statistics were performed with SAS. Results: Sixty-six different MCPV were detected. Of 111 patients, 70 (63%) with available MCPV produced at least 1 MHC I neopeptide binding. Frequently observed variants were P53 (13%), FLT3 (9.2%), WT1 (7%), DNMT3A (6.5%), RAS (5%), ASXL1 (4.3%), RUNX1 (4.2%), NPM1 (4.2%) and IDH1/2 (2.6%). Cluster 2 demonstrated a lack of favorable and higher proportion of adverse ELN 2022 subgroups. Cluster 3 exhibited a higher of P53 [p=0.03] and MDS-related mutations. OS was 79, 239, and 1551 days for cluster 2,1 and 3, respectively [p=0.0001]. EFS was 134, 363 and 451 days for cluster 2,1 and 3, respectively [p=0.0001]. Interestingly, Cluster 2 and 3 assembled the highest probability for peptide binding [1 v 2 v 3 = 1.76% v 36.6% v 62%, p=0.01]. Lastly, albeit in low frequencies, higher diversity of DNA repairing enzymes was seen in cluster 2. Conclusions: Different MCPV arising in AML genome are variably immunogenic and leukemia-related neopeptide binding’s ability is differentially expressed in our 3 clusters. Frequent neopeptide bindings in clusters 2 and 3 may reflect higher AML immunogenicity resulting in genomic instability. It is possible that less “edited” genomes in cluster 1 preserve higher chemosensitivity resulting in better OS, while highly edited genomes in cluster 2 and 3 are less chemo-sensitive and circumvent host immunosurveillance. Further expansion of cluster 2 is necessary to better understand the role of DNA repairing enzymes in younger patients’ outcomes, especially when genomic instability is observed. Citation Format: Paul Sackstein, Sarah Mudra, Lacey Williams, Garrett Diltz, Rachel Zemel, Sravanti Teegavarapu, Martha Mims, Catherine Lai, Kimberley Doucette, Anne Renteria, Olga Timofeeva, Gustavo A. Rivero. Investigating human leukocyte antigen (HLA) class 1 leukemogenic pathways in acute myelogenous leukemia [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 5377.

Metabolic instruction of the graft-versus-leukemia immunity.

Allogeneic hematopoietic cell transplantation (allo-HCT) is frequently performed to cure hematological malignancies, such as acute myeloid leukemia (AML), through the graft-versus-leukemia (GVL) effect. In this immunological process, donor immune cells eliminate residual cancer cells in the patient and exert tumor control through immunosurveillance. However, GVL failure and subsequent leukemia relapse are frequent and associated with a dismal prognosis. A better understanding of the mechanisms underlying AML immune evasion is essential for developing novel therapeutic strategies to boost the GVL effect. Cellular metabolism has emerged as an essential regulator of survival and cell fate for both cancer and immune cells. Leukemia and T cells utilize specific metabolic programs, including the orchestrated use of glucose, amino acids, and fatty acids, to support their growth and function. Besides regulating cell-intrinsic processes, metabolism shapes the extracellular environment and plays an important role in cell-cell communication. This review focuses on recent advances in the understanding of how metabolism might affect the anti-leukemia immune response. First, we provide a general overview of the mechanisms of immune escape after allo-HCT and an introduction to leukemia and T cell metabolism. Further, we discuss how leukemia and myeloid cell metabolism contribute to an altered microenvironment that impairs T cell function. Next, we review the literature linking metabolic processes in AML cells with their inhibitory checkpoint ligand expression. Finally, we focus on recent findings concerning the role of systemic metabolism in sustained GVL efficacy. While the majority of evidence in the field still stems from basic and preclinical studies, we discuss translational findings and propose further avenues for bridging the gap between bench and bedside.

LSD1 Inhibition Enhances Antigen Presentation and Co-Stimulation in AML to Promote T Cell-Mediated Anti-Leukemia Immune Responses

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can provide a curative treatment option for acute myeloid leukemia (AML). Donor T cells play an important role in the curative potential of allo-HSCT by recognizing and eliminating residual leukemic cells, inducing the desirable graft-versus-leukemia (GVL) effect. Evading from this effect of donor T cell immune surveillance through the downregulation of human leukocyte antigen class II (HLA-II) molecules has been shown to contribute to AML relapse after allo-HSCT. Lysine-specific demethylase 1 (LSD1) is an emerging epigenetic therapeutic target in AML due to its role in regulating myeloid differentiation pathways. We hypothesized that LSD1 inhibition has the potential to enhance anti-leukemic immune responses by differentiating AML cells and inducing the acquisition of markers typical for antigen-presenting cells. We have studied these effects by treating human AML cell lines with the LSD1 inhibitor bomedemstat and investigated the expression of antigen-presenting molecules by flow cytometry and quantitative PCR (qPCR). At a concentration of 100 nM, bomedemstat upregulated the expression of CIITA by a log2 fold change of 2.23 ± 0.68 (p < 0.01) in OCI-AML3, 2.17 ± 0.28 (p < 0.01) in HL60 and 1.80 ± 0.67 (p < 0.05) in MOLM-13. To assess if the observed upregulation of CIITA can lead to a re-expression of HLA-DR in these HLA-DR negative cell lines, we then treated these cell lines with 100 nM of bomedemstat with additional IFN-γ stimulation. Expression of HLA-DR and the co-stimulatory molecule CD86 was determined by flow cytometry. Bomedemstat increased the expression of HLA-DR and CD86 across all three cell lines leading to the generation of 26.63 ± 3.07% (p < 0.0001) HLA-DR +/CD86 + cells in OCI-AML3, 4.19 ± 1.68% (p < 0.001) HLA-DR +/CD86 + cells in HL-60 and 14.67 ± 2.03% (p < 0.001) HLA-DR +/CD86 + cells in MOLM-13. We further assessed the effects of bomedemstat treatment on cytokine production by qPCR. Notably, bomedemstat treatment increased the expression of CXCL-10 by a log2 fold change of 2.59 ± 0.66 (p < 0.001) in MOLM-13 and 4.12 ± 0.88 (p < 0.001) in HL-60 at 100 nM concentration. The upregulation of CXCL-10 was also validated using bead-based ELISA assays. To investigate the molecular mechanism of the observed concomitant upregulation of MHC-II molecules and CD86, we then used murine myeloid progenitor cells transformed by overexpression of Hoxa9 and Meis1 (H9M) or MN1. In H9M-transformed cells, bomedemstat concurrently upregulated MHC-II (I-A/I-E) and CD86 even in the absence of IFN-γ stimulation, leading to an increase in MHC-II +/CD86 + cells from 2.52 ± 1.22% at baseline to 29.95 ± 14.11 (p < 0.05). This effect was not observed in MN1-transformed cells. H9M-transformed progenitor cells from Irf8 knockout mice (H9M- Irf8-KO) did not show any upregulation of MHC-II, pointing towards a role for Irf8 in this process. To interrogate whether the concurrent upregulation of MHC-II and CD86 can enhance CD4 + T cell activation, we used ovalbumin-expressing H9M-transformed cells that were co-cultured with OT-II T cells after bomedemstat treatment. We could show that bomedemstat-treatment enhanced the ability of H9M-OVA cells to activate T cells as demonstrated by an enhanced expression of the T cell activation markers CD25 and CD69. Furthermore, bomedemstat also increased the immune killing of OVA-peptide pulsed H9M cells at an effector-to-target (E:T) ratio of 1:1 from 1.12 ± 4.01 to 19.70 ± 6.20% (p < 0.01). It also enhanced the killing of OVA-expressing H9M cells from 5.80 ± 2.95% to 17.70 ± 3.16% (p < 0.05). No significant killing effect was observed for MN1 cells. In conclusion, we demonstrate both phenotypically and functionally that LSD1 inhibition by bomedemstat can enhance the immunogenicity of human and murine AML models. These findings point towards a potential role of LSD1 inhibition as a maintenance therapy after allo-HSCT.

Single-cell analysis of immune recognition in chronic myeloid leukemia patients following tyrosine kinase inhibitor discontinuation

Immunological control of residual leukemia cells is thought to occur in patients with chronic myeloid leukemia (CML) that maintain treatment-free remission (TFR) following tyrosine kinase inhibitor (TKI) discontinuation. To study this, we analyzed 55 single-cell RNA and T cell receptor (TCR) sequenced samples (scRNA+TCRαβ-seq) from patients with CML (n = 13, N = 25), other cancers (n = 28), and healthy (n = 7). The high number and active phenotype of natural killer (NK) cells in CML separated them from healthy and other cancers. Most NK cells in CML belonged to the active CD56dim cluster with high expression of GZMA/B, PRF1, CCL3/4, and IFNG, with interactions with leukemic cells via inhibitory LGALS9–TIM3 and PVR–TIGIT interactions. Accordingly, upregulation of LGALS9 was observed in CML target cells and TIM3 in NK cells when co-cultured together. Additionally, we created a classifier to identify TCRs targeting leukemia-associated antigen PR1 and quantified anti-PR1 T cells in 90 CML and 786 healthy TCRβ-sequenced samples. Anti-PR1 T cells were more prevalent in CML, enriched in bone marrow samples, and enriched in the mature, cytotoxic CD8 + TEMRA cluster, especially in a patient maintaining TFR. Our results highlight the role of NK cells and anti-PR1 T cells in anti-leukemic immune responses in CML.

Coordinated Immune Cell Networks in the Bone Marrow Microenvironment Define the Graft Versus Leukemia Response with Adoptive Cellular Therapy

Donor lymphocyte infusion (DLI) is an established therapy for relapsed acute myeloid leukemia (AML) after hematopoietic stem cell transplant (HSCT), but response rates are poor (~20%). Interactions between leukemia and immune cells within the leukemia microenvironment may determine responsiveness to adoptive cellular immunotherapies. We hypothesized that systematic characterization of the leukemic marrow microenvironment over treatment course with DLI would define leukemia-immune cell interactions critical to response and hence provide mechanistic understanding of the graft-versus-leukemia (GvL) effect. In particular, we anticipated that use of multi-modal single-cell sequencing across patients and time points before and after therapy accounting for temporal dependencies would facilitate disentangling of the complex microenvironment of leukemic marrow. To infer cell-cell interactions in a temporally resolved fashion from single cell RNA-sequencing (scRNA-seq) data, we developed DIISCO, a Bayesian model using a Gaussian process regression network. In total, we profiled 30 bone marrow aspirates (14 pre- and 16 post-DLI) from 9 patients (5 responder [R], 4 nonresponder [NR]) with post-HSCT relapsed AML treated with DLI by scRNA-seq, single cell TCR sequencing (scTCR-seq) and surface protein characterization (scCITE-seq). We obtained 51,371 high-quality transcriptomes from the marrow samples along with 43,918 cells from the DLI products of these patients (4 R, 3 NR) for a total of 95,289 cells. Cells were clustered with Phenograph resulting in 57 clusters, including 2 CD4+ T cell, 6 CD8+ T cell (C0, C5, C21, C25, C26, C40), 3 NK, 10 B cell, 3 AML leukemia, and several myelo-erythroid clusters. DIISCO revealed a cascading response post-DLI that centered around CD8 C0, CD8 C5, AML, and a B cell cluster in R, not observed in NR. Furthermore, C0 appeared to expand after DLI only in R. We observed a strong negative interaction from CD8 C0 to AML after DLI, suggesting an anti-leukemia immune response, supporting the notion that immune cell populations formed a coordinated interactome associated with effective GvL. Evaluation of inferred interacting pairs using DIISCO and with the tool CellPhoneDB pointed toward an activation circuit in R between CD226 on CD8 C0 and PVR/NECTIN2 on AML. Conversely, inhibitory interactions between the exhaustion marker TIGIT on CD8 C0 and AML were observed in NR. Because DIISCO pinpointed CD8 C0 as a central hub for response, we characterized the identity of this cluster and its post-DLI dynamics. scTCR-seq revealed that this cluster, but not other T cell clusters, underwent marked clonal expansion after DLI ( Figure 1A). scRNA-seq revealed high expression of activation/effector genes ( GZMB, IFNG, CX3CR1, PRF1) compared to other T cells. The clonally expanded cells of C0 also highly expressed ZNF683, a transcription factor associated with T cell cytotoxicity. scCITE-seq demonstrated expression of CD45RA and CD57 but not CD28, consistent with an effector memory re-expressing RA (TEMRA) phenotype. We validated post-DLI expansion of CD8 TEMRA cells (CD3+, CD8+, CD62L-, CD45RA+) in AML R by multi-parameter flow cytometry in an independent cohort of 53 AML patients (29 R, 24 NR) with post-HSCT relapse treated with DLI at our institution (fold change 2.56 pre to post for R, p<0.001; 1.3 pre to post for NR, p=0.474). Moreover, to examine whether CD8+ TEMRA cells were specifically activated after encounter with leukemia, we devised an in vitromodified mixed lymphocyte reaction. For 6 AML R, post-DLI donor-derived CD8+ TEMRA cells that were cultured with pre-DLI recipient leukemia cells consistently demonstrated greater CD137 (p=0.004) and IFN-γ secretion compared to co-culture of the leukemia cells with other T cell subsets (p=0.015) ( Figure 1B). CD8+ TEMRA cells cultured in the absence of leukemia cells did not show elevated expression of either CD137 or IFN-γ, indicating antigen-specific functional activation of CD8+ TEMRAs but not other cell types after encounter with autologous leukemia. Our results reveal clonal expansion and tumor-specific activation of CD8+ TEMRA cells, supporting an antigen specific functional role of these cells in mediating GvL in AML DLI responders. Further evaluation of this CD8 TEMRA population with the ability to recognize leukemia cells may translate to strategies to optimize adoptive cellular therapy or predict responsiveness to DLI.

Volatile Phases Derived from Serum, DC, or MLC Culture Supernatants to Deduce a VOC-Based Diagnostic Profiling Strategy for Leukemic Diseases.

Volatile organic compounds (VOCs) reflect the metabolism in healthy and pathological conditions, and can be collected easily in a noninvasive manner. They are directly measured using electronical nose (eNose), and may qualify as a systemic tool to monitor biomarkers related to disease. Myeloid leukemic blasts can be transformed into leukemia-derived dendritic cells (DCleu) able to improve (anti-leukemic) immune responses. To profile immunological changes in healthy and acute myeloid leukemic (AML) patients' ex vivo cell cultures, we correlated the cell biological data with the profiles of cell culture supernatant-derived VOCs. DC/DCleu from leukemic or healthy whole blood (WB) were generated without (Control) or with immunomodulatory Kit M (Granulocyte macrophage-colony-stimulating-factor (GM-CSF) + prostaglandin E1 (PGE1)) in dendritic cell cultures (DC culture). Kit-pretreated/not pretreated WB was used to stimulate T cell-enriched immunoreactive cells in mixed lymphocyte cultures (MLC culture). Leukemia-specific adaptive and innate immune cells were detected with a degranulation assay (Deg) and an intracellular cytokine assay (InCyt). Anti-leukemic cytotoxicity was explored with a cytotoxicity fluorolysis assay (CTX). VOCs collected from serum or DC- and MLC culture supernatants (with vs. without Kit M pretreatment and before vs. after culture) were measured using eNose. Compared to the Control (without treatment), Kit M-pretreated leukemic and healthy WB gave rise to higher frequencies of mature (leukemia-derived) DC subtypes of activated and (memory) T cells after MLC. Moreover, antigen (leukemia)-specific cells of several lines (innate and adaptive immunity cells) were induced, giving rise to blast-lysing cells. The eNose could significantly distinguish between healthy and leukemic patients' serum, DC and MLC culture supernatant-derived volatile phases and could significantly separate several supernatant (with vs. without Kit M treatment, cultured vs. uncultured)-derived VOCs within subgroups (healthy DC or leukemic DC, or healthy MLC or leukemic MLC supernatants). Interestingly, the eNose could indicate a Kit M- and culture-associated effect. The eNose may be a prospective option for the deduction of a VOC-based profiling strategy using serum or cell culture supernatants and could be a useful diagnostic tool to recognize or qualify AML disease.

B cell leukemia elicits a suppressive Tr1-like CD4 T cell response that can be reversed with anti-PDL1-blockade

Abstract CD4 T cell exhaustion is associated with relapse in B cell leukemia (B-ALL) patients. However, checkpoint blockade is not effective in treating B-ALL. Using a murine model of B-ALL, we demonstrate that leukemia induced CD4 T cells had a unique regulatory-cytotoxic phenotype (T rctx) with preferential expression of exhaustion markers (PD1/TIM3) as well as IL10 and c-Maf, which are markers of immunosuppressive Tr1 cells. Leukemia cells acting as APCs elicited CD4 T cells with a similar Tr1 phenotype that suppressed bystander T cells. This process mimics the mechanism by which hematopoietic stem cells (HSC) enforce an immunosuppressive niche in the bone marrow that protects the HSC compartment. Anti-PDL1 ornilotinib (targeted therapy) treatment alone had a minimal impact on survival; concurrent treatment with anti-PDL1 andnilotinib had a significant synergistic effect that was dependent on CD4 and CD8 T cells. Nilotinib treatment increased markers of helper and regulatory function in the T rctxcells. In contrast, treatment with nilotinib andanti-PDL1 induced expansion of a leukemia-specific CD4 T cell clone with lower expression of Tr1 markers and higher expression of helper molecules that facilitate recruitment and activation of anti-tumor immune cells. Expansion of the helper-cytotoxic (T hctx) CD4 T cell clone was associated with increased GzmB +CD8 T cells. We propose that leukemia coopts a naturally occurring process designed to protect the HSC niche, thereby inducing T rctxcells that drive escape from immune surveillance. Combining nilotinib and anti-PDL1 alters the balance between immunosuppressive T rctxand immune-activating T hctx, thereby inducing a protective anti-leukemia immune response that prevents relapse. Spatial Transcriptomics Award from the University of Minnesota, Children's Cancer Research Fund

Engineered murine IL-21-secreting leukemia cells induce granzyme B+ T cells and CD4+CD44+CD62L- effector memory cells while suppressing regulatory T cells, leading to long-term survival.

We have explored the use of an IL-21 cell-based anti-leukemia treatment in a mouse model of acute lymphoblastic leukemia. 70Z/3 leukemia cells, engineered to secrete IL-21 and injected into the peritoneum of syngeneic mice, induced a strong anti-leukemia response resulting in 100% survival. Mice that mounted an IL-21-induced anti-leukemia immune response were immune to the parent cell line (no IL-21) when rechallenged.Above a certain threshold, IL-21 secretion correlated with improved survival compared to mice injected with parent 70Z/3 cells. IL-21 was detected in serum with peak levels on day 7, correlating with the maximum expansion of IL-21-secreting 70Z/3 cells which subsequently were eliminated. Mice injected with IL-21-secreting leukemia cells had elevated numbers of granzyme B+ CD4+ and CD8+ T cells in the peritoneum, compared to mice injected with the parent cell line. Regulatory T cells, which increased greatly in 70Z/3-injected mice, failed to do so in mice injected with IL-21-secreting cells. Upon rechallenge, IL-21-primed mice went through a secondary immune response, primarily requiring CD4+ T cells, triggering a significant increase of CD4+CD44+CD62L- effector memory T cells. Adoptive transfer of T cells from IL21-primed/rechallenged hosts into naïve mice was successful, indicating that IL-21-primed antigen-experienced T cells convey immunity to naïve mice.Our study shows that delivery of IL-21 in a cell-based anti-leukemia protocol has the potential to induce a potent immune response leading to cancer elimination and long-term immunity-properties which make IL-21 an attractive candidate for cancer immunotherapy. Protecting against tumor antigens as well as improving cancer immunity is justified, as current strategies are limited.

Myelodysplastic Syndrome associated TET2 mutations affect NK cell function and genome methylation

Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders, representing high risk of progression to acute myeloid leukaemia, and frequently associated to somatic mutations, notably in the epigenetic regulator TET2. Natural Killer (NK) cells play a role in the anti-leukemic immune response via their cytolytic activity. Here we show that patients with MDS clones harbouring mutations in the TET2 gene are characterised by phenotypic defects in their circulating NK cells. Remarkably, NK cells and MDS clones from the same patient share the TET2 genotype, and the NK cells are characterised by increased methylation of genomic DNA and reduced expression of Killer Immunoglobulin-like receptors (KIR), perforin, and TNF-α. In vitro inhibition of TET2 in NK cells of healthy donors reduces their cytotoxicity, supporting its critical role in NK cell function. Conversely, NK cells from patients treated with azacytidine (#NCT02985190; https://clinicaltrials.gov/) show increased KIR and cytolytic protein expression, and IFN-γ production. Altogether, our findings show that, in addition to their oncogenic consequences in the myeloid cell subsets, TET2 mutations contribute to repressing NK-cell function in MDS patients.

Amplifying STING activation by bioinspired nanomedicine for targeted chemo- and immunotherapy of acute myeloid leukemia.

Chemotherapy resistance and the tumor immune microenvironment are dual reasons for the poor therapeutic efficacy of treating acute myeloid leukemia (AML), causing suboptimal clinical outcomes and high relapse rates. Activation of the stimulator of interferon genes (STING) pathway based on innate immunity can effectively improve antitumor immunity. However, traditional STING agonists are limited due to their easy degradation and difficult membrane transport. Here, a bioinspired nanomedicine synergizing chemo- and immunotherapy was developed by activating the STING pathway for targeted and systemic AML cell damage. We show that a leukemia cell membrane (LCM)-camouflaged hollow MnO2 nanocarrier (HM) with encapsulated doxorubicin (DOX) (denoted LHMD) could bind specifically to AML cells with a homologous targeting effect. Then, MnO2 was decomposed into Mn2+ in response to endosomal acid and glutathione (GSH), which improved the magnetic resonance imaging (MRI) signal for AML detection and activated the STING pathway. In mouse models, LHMD was confirmed to eradicate established AML and prevent the engraftment of AML cells. The percentages of T-helper 1 (Th1) and T-helper 17 (Th17) cells and the concentrations of type I interferon (IFN-Ⅰ) and proinflammatory cytokines increased, while the percentage of T-helper 2 (Th2) cells decreased, reflecting the anti-AML immune response induced by Mn2+ after treatment with LHMD. This nanotechnology-based therapeutic regimen may represent a generalizable strategy for generating an anti-leukemia immune response. STATEMENT OF SIGNIFICANCE: Relapse and chemotherapy refractoriness are main causes for the dismal prognosis of AML, making it urgent to develop more effective anti-AML therapies. This study proposes an innovative strategy to combat this issue by designing a biomimetic BM-targeted nanomedicine based on a MnO2 nano-carrier to rationally deliver chemotherapeutic agents and to trigger Mn2+ mediated STING pathway activation for potent immune- and chemotherapy against AML cells. Hence, the nanomedicine design addresses the challenges associated with AML therapy and proposes a promising strategy to improve the therapeutic efficacy against AML.

Abstract 240: MERTK inhibition induces an anti-leukemia dendritic cell - T cell axis while TYRO3 inhibition protects through a separate mechanism

Abstract The TAM family receptor tyrosine kinases TYRO3, AXL and MERTK are potential therapeutic targets in a variety of cancers. In previous studies, inhibition of MERTK decreased PD-1 checkpoint proteins in the leukemia microenvironment and prolonged survival in a syngeneic BCR-ABL+/Arf-/- B-cell acute leukemia model, implicating MERTK as a promising immune-oncology target in leukemia. Strikingly, Mertk-/- mice were largely protected from leukemia. In our current studies, Tyro3-/- almost completely prevented development of leukemia, comparable to Mertk-/-, while Axl-/- mice died with similar timing to wild type (WT) mice (20-40 days). These data demonstrate differential roles for TAM kinases in the anti-leukemia immune response. Depletion studies were conducted to evaluate potential roles for T cells and dendritic cells (DCs) in anti-leukemia immunity in Mertk-/- mice. Selective depletion of CD8+ T cells abrogated protection from leukemia in Mertk-/- mice, but survival was still prolonged relative to WT. Thus, while CD8+ T cells were required for complete protection from leukemia, the anti-leukemia response remained partially intact even in the absence of CD8+ T cells, implicating an innate immune mechanism. Indeed, combined depletion of CD8+ T cell and CD8α+ DC subsets completely abrogated the anti-leukemic effects in Mertk-/- mice, revealing a critical immunosuppressive role for MERTK in DCs in the leukemia microenvironment. In contrast to Mertk-/- mice, selective depletion of CD8+ T cells completely abrogated protection from leukemia in Tyro3-/- mice, indicating a mechanism less dependent on DCs. Similarly, single cell RNA sequencing revealed CD8+ DCs with a more mature and antigen-presenting phenotype in Mertk-/- mice compared to WT, while antigen-presenting DCs were not increased in Tyro3-/- mice. Single cell sequencing data also suggest induction of an anti-leukemic DC - T cell axis in WT leukemic mice treated with the MERTK-selective inhibitor MRX-2843. DCs were nearly absent in leukemic bone marrow from saline-treated mice and were dramatically increased in response to treatment with MRX-2843. Treatment with MRX-2843 also decreased the incidence of CD8+ T cells expressing high levels of Tox, which has been associated with T cell exhaustion. These changes coincided with decreased leukemic blasts, even in the context of established disease.Together, our findings support a model whereby MERTK inhibition promotes DC function and CD8+ T cell activity, leading to anti-leukemia immunity. In contrast, anti-leukemia immunity in response to TYRO3 inhibition is less dependent on DCs. Differential roles for the TAM kinases in the leukemia microenvironment provide rationale for development of MERTK and/or TYRO3 targeted immunotherapies to treat acute leukemia. Citation Format: Justus M. Huelse, Swati S. Bhasin, Beena E. Thomas, Madison L. Chimenti, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Manoj Bhasin, Deborah DeRyckere, Douglas K. Graham. MERTK inhibition induces an anti-leukemia dendritic cell - T cell axis while TYRO3 inhibition protects through a separate mechanism [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 240.

NPM1 and DNMT3A mutations are associated with distinct blast immunophenotype in acute myeloid leukemia

ABSTRACT The immune system is important for elimination of residual leukemic cells during acute myeloid leukemia (AML) therapy. Anti-leukemia immune response can be inhibited by various mechanisms leading to immune evasion and disease relapse. Selected markers of immune escape were analyzed on AML cells from leukapheresis at diagnosis (N = 53). Hierarchical clustering of AML immunophenotypes yielded distinct genetic clusters. In the absence of DNMT3A mutation, NPM1 mutation was associated with decreased HLA expression and low levels of other markers (CLIP, PD-L1, TIM-3). Analysis of an independent cohort confirmed decreased levels of HLA transcripts in patients with NPM1 mutation. Samples with combined NPM1 and DNMT3A mutations had high CLIP surface amount suggesting reduced antigen presentation. TIM-3 transcript correlated not only with TIM-3 surface protein but also with CLIP and PD-L1. In our cohort, high levels of TIM-3/PD-L1/CLIP were associated with lower survival. Our results suggest that AML genotype is related to blast immunophenotype, and that high TIM-3 transcript levels in AML blasts could be a marker of immune escape. Cellular pathways regulating resistance to the immune system might contribute to the predicted response to standard therapy of patients in specific AML subgroups and should be targeted to improve AML treatment.

Combining nilotinib and PD-L1 blockade reverses CD4+ T-cell dysfunction and prevents relapse in acute B-cell leukemia

AbstractPatients with acute lymphoblastic leukemia have experienced significantly improved outcomes due to the advent of chimeric antigen receptor (CAR) T cells and bispecific T-cell engagers, although a proportion of patients still relapse despite these advances. T-cell exhaustion has been recently suggested to be an important driver of relapse in these patients. Indeed, phenotypic exhaustion of CD4+ T cells is predictive of relapse and poor overall survival in B-cell acute lymphoblastic leukemia (B-ALL). Thus, therapies that counter T-cell exhaustion, such as immune checkpoint blockade, may improve leukemia immunosurveillance and prevent relapse. Here, we used a murine model of Ph+ B-ALL as well as human bone marrow biopsy samples to assess the fundamental nature of CD4+ T-cell exhaustion and the preclinical therapeutic potential for combining anti–PD-L1 based checkpoint blockade with tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein. Single-cell RNA-sequence analysis revealed that B-ALL induces a unique subset of CD4+ T cells with both cytotoxic and helper functions. Combination treatment with the tyrosine kinase inhibitor nilotinib and anti–PD-L1 dramatically improves long-term survival of leukemic mice. Depletion of CD4+ T cells prior to therapy completely abrogates the survival benefit, implicating CD4+ T cells as key drivers of the protective anti-leukemia immune response. Indeed, treatment with anti–PD-L1 leads to clonal expansion of leukemia-specific CD4+ T cells with the aforementioned helper/cytotoxic phenotype as well as reduced expression of exhaustion markers. These findings support efforts to use PD1/PD-L1 checkpoint blockade in clinical trials and highlight the importance of CD4+ T-cell dysfunction in limiting the endogenous anti-leukemia response.

A Specific CD44lo CD25lo Subpopulation of Regulatory T Cells Inhibits Anti-Leukemic Immune Response and Promotes the Progression in a Mouse Model of Chronic Lymphocytic Leukemia.

Regulatory T cells (Tregs) are capable of inhibiting the proliferation, activation and function of T cells and play an important role in impeding the immune response to cancer. In chronic lymphocytic leukemia (CLL) a dysfunctional immune response and elevated percentage of effector-like phenotype Tregs have been described. In this study, using the Eµ-TCL1 mouse model of CLL, we evaluated the changes in the Tregs phenotype and their expansion at different stages of leukemia progression. Importantly, we show that Tregs depletion in DEREG mice triggered the expansion of new anti-leukemic cytotoxic T cell clones leading to leukemia eradication. In TCL1 leukemia-bearing mice we identified and characterized a specific Tregs subpopulation, the phenotype of which suggests its role in the formation of an immunosuppressive microenvironment, supportive for leukemia survival and proliferation. This observation was also confirmed by the gene expression profile analysis of these TCL1-specific Tregs. The obtained data on Tregs are consistent with those described so far, however, above all show that the changes in the Tregs phenotype described in CLL result from the formation of a specific, described in this study Tregs subpopulation. In addition, functional tests revealed the ability of Tregs to inhibit T cells that recognize model antigens expressed by leukemic cells. Moreover, inhibition of Tregs with a MALT1 inhibitor provided a therapeutic benefit, both as monotherapy and also when combined with an immune checkpoint inhibitor. Altogether, activation of Tregs appears to be crucial for CLL progression.

The Importance of Cellular Metabolic Pathways in Pathogenesis and Selective Treatments of Hematological Malignancies.

Despite recent advancements in the treatment of hematologic malignancies and the emergence of newer and more sophisticated therapeutic approaches such as immunotherapy, long-term overall survival remains unsatisfactory. Metabolic alteration, as an important hallmark of cancer cells, not only contributes to the malignant transformation of cells, but also promotes tumor progression and metastasis. As an immune-escape mechanism, the metabolic adaptation of the bone marrow microenvironment and leukemic cells is a major player in the suppression of anti-leukemia immune responses. Therefore, metabolic rewiring in leukemia would provide promising opportunities for newer therapeutic interventions. Several therapeutic agents which affect essential bioenergetic pathways in cancer cells including glycolysis, β-oxidation of fatty acids and Krebs cycle, or anabolic pathways such as lipid biosynthesis and pentose phosphate pathway, are being tested in various types of cancers. So far, numerous preclinical or clinical trial studies using such metabolic agents alone or in combination with other remedies such as immunotherapy are in progress and have demonstrated promising outcomes. In this review, we aim to argue the importance of metabolic alterations and bioenergetic pathways in different types of leukemia and their vital roles in disease development. Designing treatments based on targeting leukemic cells vulnerabilities, particularly in nonresponsive leukemia patients, should be warranted.

Overcoming Leukemia-Induced Immune Suppression in BCR-ABL+ Acute Lymphoblastic Leukemia

BCR-ABL+ acute lymphoblastic leukemia patients have transient responses to current therapies. However, the fusion of BCR to ABL generates a potential leukemia-specific antigen that could be a target for immunotherapy. Herein we demonstrate that the immune system can limit BCR-ABL+ leukemia progression although ultimately this immune response fails. To address how BCR-ABL+ leukemia escapes immune surveillance, we developed a peptide: MHC-II tetramer that labels endogenous BCR-ABL-specific CD4+ T cells. Naïve mice have a small population of BCR-ABL-specific T cells that proliferate modestly upon immunization with the BCR-ABL fusion peptide (referred to as BAp hereafter). In response to BCR-ABL+ leukemia, BCR-ABL specific T cells proliferated and converted into regulatory T cells (Treg cells), a process that was dependent on MHC-II antigen presentation by leukemic cells. Treg cells were critical for leukemia progression in C57Bl/6 mice, as transient Treg cell ablation led to extended survival of leukemic mice. Leukemic B cells produced significantly more TGb1, IL10 and PDL1 than control non-transformed B cells. We are currently extending these studies to determine if leukemia-specific production of TGFb, IL10, or PDL1 plays a critical role in driving tolerance induction. Thus, BCR-ABL+ leukemia actively suppresses the anti-leukemia immune responses by converting cross-reactive leukemia-specific T cells into Treg cells. To determine if we could reverse tolerance we examined whether checkpoint blockade or therapeutic vaccination with BAp peptide could improve survival in mice with pre-established BCR-ABL+ leukemia. Consistent with the low mutation load in our leukemia model, we found that checkpoint blockade with anti-PDL1 and CTLA-4 alone had only modest effects on survival. In contrast, robust heterologous vaccination using LCMV, Listeria, and VSV infection plus a peptide derived from the BCR-ABL fusion (BAp), a key driver mutation, resulted in a small population of mice that survived long-term. Checkpoint blockade strongly synergized with heterologous vaccination to enhance overall survival in mice with leukemia. Enhanced survival did not correlate with numbers of BAp:I-Ab-specific T cells, but rather with increased expression of IL10, IL17, and GrzmB and decreased expression of PD1 on these cells. Our findings demonstrate that vaccination to key driver mutations cooperates with checkpoint blockade and allows for immune control of cancers with low non-synonymous mutation loads. We are currently examining whether we can further improve survival via combination chemotherapy (using Nilotinib which typically only induces transient remission) and immunotherapy (using checkpoint blockade with anti-PD1 or anti-PDL1). DisclosuresFarrar:Merck: Research Funding.

Bone Marrow CD8 T Cells Express High Frequency of PD-1 and Exhibit Reduced Anti-Leukemia Response in AML Patients

Acute myeloid leukemia (AML) is a devastating blood cancer with 5- year survival of only 25%. Optimizing anti-leukemia immune response to improve clinical outcome is a promising strategy for novel effective leukemia therapeutics. However due to limited accessibility, the majority of studies have been restricted to evaluate samples from peripheral blood. The immune response in bone marrow of AML is poorly understood. In this study, we aim to investigate the T cell immune response in bone marrow samples derived from AML patients.Samples collected from a cohort of patients with newly diagnosed AML (n=19) were used in this study. Mononuclear cells of bone marrow and peripheral blood were isolated from clinical samples before multi-channel flow cytometry analysis. We observed a comparable frequency of CD3+ T cells among lymphocytes in bone marrow and peripheral blood. Similarly there is no difference of CD4, CD8 T cell frequency, or CD4/CD8 ratio. However, when T cell subsets for each differentiation stage were analyzed based on the expression of CD45RA and CCR7, we found significantly increased effector memory T cells (CD45RA-CCR7-) among CD8 T cells in bone marrow compared with that in peripheral blood (43.88±5.012% vs. 35.15±5.104%, P =0.0088). Whereas the percentage of terminally differentiated effector cells (CD45RA+CCR7-) was significantly lower in bone marrow (20.36±5.025% vs. 28±5.947%, P =0.0481). No difference was found in the frequency of naive (CD45RA+CCR7+) and central memory (CD45RA-CCR7+) subsets.Several studies including ours have demonstrated an involvement of the inhibitory receptors PD-1, TIM-3 and TIGIT in AML progression. Most of the studies were performed using clinical samples from peripheral blood. To evaluate whether the effect of inhibitory pathways is different between bone marrow and peripheral blood, we assessed the expression of these inhibitory receptors on T cells derived from both sites. We observed comparable expression of TIGIT and TIM-3 in bone marrow and peripheral blood. The frequency of PD-1 expression on CD8 T cells was however significantly higher in bone marrow (39.55±3.809% vs. 32.8±3.512%, P =0.0058). We further dissected the intracellular expression of Eomesodermin (Eomes) and T-bet, the two T box transcription factors that are crucial in regulating T cell function. We found that the frequency of Eomes+T-betint CD8 T cells was significantly higher in bone marrow than in peripheral blood (17.01±1.843% vs. 14.95±1.682%, P =0.0405), indicating a predominance for late stage of T cell exhaustion status in bone marrow. This result suggests a relatively more suppressive environment in bone marrow compared with that of peripheral blood.We next examined the functional status of CD8 T cells. Intracellular IFN-γ and TNF-α production was assessed upon in vitro stimulation with anti-CD3/CD28. Intriguingly no significant difference between bone marrow and peripheral blood was detected. Consistently we observed no difference between the two sites in term of the expression of Ki67, Granzyme B, and Perforin, suggesting a comparable proliferation and killing capacity of CD8 T cells between bone marrow and peripheral blood. We further investigated the function of leukemia-reactive CD8 T cells by testing T cell response against a WT-1 peptide. WT-1 is a well known tumor-associated antigen, in which HLA-A*0201 restricted WT-1126-134 is one of the mostly studied epitope in AML. Purified CD8 T cells derived from bone marrow or blood of newly diagnosed HLA-A*0201 AML patients were co-cultured with T2 cells (used as antigen presenting cells) pulsed with WT-1126-134. We found a significantly lower production of IFN- γ by CD8 T cells from bone marrow compared with that from peripheral blood (0.4325±0.1612% vs, 0.7425±0.2809%, P =0.0472). These data demonstrate that although the total CD8 T cells in bone marrow remain functional, the leukemia-reactive CD8 T cells are less functional in bone marrow compared with that in peripheral blood.Collectively, our data demonstrate that bone marrow CD8T cells from AML patients express higher frequency of PD-1 and exhibit reduced anti-leukemia response. Our study suggests that bone marrow is a unique anatomical location fostering negative immune response in AML, thus targeting the negative regulatory pathways in bone marrow is of great potential to develop novel effective leukemia treatment. DisclosuresNo relevant conflicts of interest to declare.