Immunotherapy Of Acute Myeloid Leukemia Research Articles

Spatial Technologies Reveal the Immune Landscape of Pediatric Acute Myeloid Leukemia

Pediatric acute myeloid leukemia (AML) is a cancer with a particularly low mutational burden in comparison to other pediatric and adult cancers and therefore, thought to be a poor candidate for T cell-engaging immunotherapies (Gröbner et al., 2018; Pfister et al., 2022). However, little is known about the composition and function of T cells in the bone marrow (BM) of pediatric AML patients. Insight into the frequency and function of BM T cells in children with AML is relevant for naturally occurring AML immune defenses, as well as for T cell-engaging immunotherapies (Koedijk et al., 2021). Here, we performed a multidimensional characterization of the tumor immune microenvironment in children with newly diagnosed AML. We obtained 82 formalin-fixed and paraffin-embedded (FFPE) diagnostic bone biopsies from a representative cohort of pediatric patients with treatment-naïve de novo AML (N=72) and from age- and sex-matched pediatric patients with treatment-naïve early-stage rhabdomyosarcoma without malignant BM infiltration (non-leukemic controls, N=10). We employed immunohistochemistry (IHC) alongside immune-related gene expression profiling (NanoString PanCancer IO 360 TM Panel) and spatial transcriptomics (NanoString GeoMx TM Whole Transcriptome Atlas). Moreover, we acquired an additional dataset of pre-treatment immune-related gene expression profiling obtained from the BM of 30 flotetuzumab-treated (CD3 x CD123 bispecific antibody) refractory/relapsed (R/R) adult AML patients (NCT02152956; Vadakekolathu et al., 2020) for TIDE-based immune deconvolution (Jiang et al., 2018). Using IHC, we identified (a trend towards) a decreased abundance of the overall number of T cells ( P=0.09) and CD8 + T cells ( P=0.011; Panel A) in the BM of pediatric AML patients in comparison to non-leukemic controls, respectively. Notably, the extent of overall T cell and CD8 + T cell infiltration could differ up to 180-fold between individual AML patients. In general, this difference in T cell infiltration was not associated with the abundance of leukemic blasts or patient's cytogenetic alterations. Nevertheless, children with complex karyotype AML had higher numbers of BM T cells compared to children with normal karyotype AML ( P=0.03).Using differential gene expression analysis, we identified genes related to T cell-attracting chemokines, cytotoxicity, and immune checkpoints to be significantly upregulated in T cell-infiltrated- compared to T cell-depleted samples. Moreover, the ratio of anti- to pro-inflammatory macrophages (M2/M1-ratio) was significantly lower in T cell-infiltrated- compared to T cell-depleted pediatric AML samples ( P<0.001). Interestingly, this M2/M1-ratio was also significantly lower in R/R adults that responded to flotetuzumab immunotherapy in comparison to non-responders ( P<0.001). In fact, this M2/M1-ratio outperformed several other known predictors of response to flotetuzumab immunotherapy (AUROC: 0.852 (95% CI: 0.71-0.99), P=0.001; Jiang et al., 2018; Ayers et al., 2017). In addition, IHC revealed that 9 immune-infiltrated samples, including 6 KMT2A-rearranged AML samples, harbored large networks of T- and B cells (representative image of B cell-networks shown in panel B). Using spatial transcriptomics, we dissected the composition of these lymphoid aggregates and revealed localized anti-tumor immunity in the BM of AML. In comparison to tumor areas, these aggregates showed a higher abundance of activated cytotoxic T cells ( P<0.001), memory B cells ( P<0.001), and plasma cells ( P<0.001), suggesting the presence of tertiary lymphoid structure-like (TLS-like) aggregates in the BM of AML. In conclusion, we identified a subset of pediatric AML patients with relatively high T cell infiltration and a relatively low abundance of anti-inflammatory macrophages in the BM at diagnosis. Furthermore, we found that the BM M2/M1-ratio may be informative of response to T-cell engaging immunotherapies in adult AML, which needs to be validated in pediatric AML. Lastly, for the first time, we identified TLS-like aggregates in the BM of AML patients, which have been associated with immunotherapy response in many cancers (Schumacher & Thommen, 2022). Additional studies to further characterize the function and relevance of these lymphoid aggregates are ongoing.

Epitope Edited Hematopoietic Stem Cells to Enable Synergistic Immunotherapy Combinations for Acute Myeloid Leukemia

Despite treatment advances, acute myeloid leukemia (AML) is still associated with an unfavorable outcome for >50% of patients. Whereas novel immunotherapies, such as CAR-T cells, bispecific and toxin conjugated antibodies (mAb), demonstrated clinical efficacy when targeting dispensable lineage antigens (Ag), such as CD19 in B-ALL, the same approach cannot be exploited for AML, due to lack of actionable leukemia-restricted Ags. Suitable targets are shared with healthy progenitor or mature myeloid cells, leading to on-target/off-tumor toxicity and impairment of hematopoietic reconstitution. Several AML immunotherapies are currently under development, but their use is restricted to a limited time window, likely insufficient for disease eradication. To address these issues, we reasoned that precise modification of the targeted epitopes in donor HSPC used in HSCT results in loss of mAb recognition, without affecting normal protein expression, regulation, and function ( Nature, accepted). Epitope editing allows targeting genes essential for leukemia survival regardless of shared expression or functional role in normal HSPC, thus minimizing the risk of tumor immune escape by Ag loss or downregulation. Cytokine receptors such as FLT3, KIT and CD123 are found in >85% of AML cases and their mutation (e.g., FLT3-ITD) or overexpression is associated with poor prognosis. We identified amino acid substitutions in the FLT3, KIT and CD123 extracellular domains that preserve physiologic surface expression, ligand-binding, kinase phosphorylation, colony-forming capacity, proliferative response, transcriptional and phospho-proteomic profile of CD34+ HSPCs but avoid detection by a therapeutic monoclonal Ab. Cells expressing these variants were resistant to CAR-T killing and did not induce CAR activation and proliferation during in vitro co-culture. We were able to introduce these mutations into CD34+ HSPCs by adenine base editing (ABE) with high efficiencies (90%, 85% and 75% for FLT3, KIT and CD123, respectively) without the need for dsDNA breaks. After xenotransplant into NBSGW mice, FLT3, KIT or CD123 epitope-editedHSPC sustained long-term multilineage hematopoiesis indicating editing and preserved functionality of HSCs. Upon treatment with FLT3 CAR-T in vivo, we observed sparing of human CD34+38- HSPCs, granulo-mono progenitors (GMP), B-cells and B-progenitors in the bone marrow of mice engrafted with FLT3-edited HSPCs compared to AAVS1 controls. Treatment with CD123 CAR-T cells showed protection of epitope-edited myeloid lineages, including granulocytes, DCs and HSPCs. We next generated advanced in vivo models with co-engraftment of human HSPCs and patient-derived AML xenografts (PDX), to demonstrate the selective resistance of epitope edited HSPCs and their progeny, while PDX were eradicated by FLT3 or CD123 CAR-T. To further enhance therapeutic efficacy, we are now exploring the combination of epitope-edited HSPCs and pharmacological agents with potential synergistic effects with CAR-T cells. FLT3 tyrosine kinase inhibitors (e.g., Crenolanib) have the potential to enforce surface expression of FLT3 by impairing its intracellular recycling, thus enhancing CAR-T mediated killing, but their use may be limited by overlapping toxicities on healthy hematopoiesis, in particular in the post-HSCT setting. To assess if epitope-editing could prevent not only CAR- but also TKI-mediated toxicities, we treated mice co-engrafted with FLT3- or AAVS1- edited HSPCs and a FLT3-ITD+ AML PDX with combinations of FLT3-CAR and a 2-week course of Crenolanib. Leukemia burden was reduced by Crenolanib alone and eradicated by FLT3-CAR-T. Critically, we observed preservation of healthy hematopoietic lineages only in mice engrafted with epitope-edited HSPCs, including the CAR+Crenolanib group, while AAVS1 controls showed overlapping off-tumor toxicity when treated with the CAR+Crenolanib combination. In conclusion, we believe that epitope edited HSPCs may not only enable safe and effective CAR-T immunotherapies for AML, but also allow their combination with pharmacological blockade of leukemia survival/proliferative pathways to achieve synthetic lethality mechanisms, while still avoiding dose-limiting toxicities. Further exploration of immunotherapy-synergistic combinations will be fundamental to improve the outcomes of difficult-to-treat high-risk AML patients.

Identifying Surface Protein Markers for AML Immunotherapy

Background: Acute Myeloid Leukemia (AML) is a hematologic malignancy characterized by clonal expansion and differentiation arrest of myeloid precursor cells in the bone marrow. It results in the accumulation of abnormal and immature myeloblasts, which impairs the production of normal blood cells, including erythrocytes, leukocytes, and platelets. Chimeric Antigen Receptor T-cell therapy (CAR-T therapy) and Antibody-Drug Conjugate (ADC) immunotherapies have shown significant success in the treatment of hematological malignancies. However, their efficacy in AML is limited due to the disease's genetic diversity, lack of uniform antigen targets and immunosuppressive microenvironment. Single-cell RNA sequencing (scRNA-seq) is a powerful technique that can be utilized for surface antigen discovery in various cell types, including immune cells and cancer cells. Methods: To identify potential AML cell-surface markers, we employed a comprehensive approach that involved the utilization of 3' single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing (RNA-seq), and enhanced whole exome sequencing (eWES). A total of 21 samples were analyzed, comprising 18 distinct AML patients and 3 healthy bone marrow aspirates, serving as controls. Results: Using enhanced whole exome sequencing (eWES) on tumor/normal paired samples, we defined the significant driver mutations within our sample cohort, notably DNMT3A (N=4, 21%), FLT3 (N=3, 16%), TET2 (N=3, 16%), KRAS (N=2, 11%), and GATA2 (N=2, 11%). Subsequently, we conducted scRNAseq on 21 samples, yielding a total of 185,106 high-quality cells, which were effectively clustered into 38 unbiased groups. Each sample demonstrated an average capture of 8862 cells, showcasing robust data acquisition. Our scRNAseq analysis revealed a diverse representation of major lymphoid and myeloid lineages, with prominent populations of AML blasts, T/NK cells, and progenitor cells. Notably, tumor clusters exhibited highest copy number variations and formed separate clusters from other non-tumor immune cells. To uncover key functional pathways, we conducted pathway enrichment analysis comparing tumor cells (N=131,196 cells) to non-tumor cells (N=53,910 cells). Our findings demonstrated a significant overrepresentation of cancer hallmarks, such as Allograft rejection (FDR= 0.000002), IL2/STAT5 signaling (FDR= 0.0000005), IFN-gamma response (FDR= 0.00000001) and apoptosis (FDR= 0.00000001) within the AML blasts, suggesting their potential involvement in AML pathogenesis. To identify AML specific markers, we developed analysis pipeline and performed an unbiased systematic search of the scRNAseq dataset for genes with unique expression patterns in tumor cells compared to the healthy bone marrow controls. Our analysis identified 302 differentially expressed genes, with 8 genes displaying relative cell and tissue specificity. Notably, among these genes, we unveiled key potential targets with cell surface specificity, including novel markers alongside previously known ones. Noteworthy candidates, such as CD86, CSF1R, CD33, CLEC12A, and FLT3, were prominently and significantly expressed, showcasing their potential significance as targets for engineered immunotherapy. To validate and consolidate our discoveries, we conducted a cross-referencing exercise by comparing our scRNAseq data against bulk RNA data from primary human AML samples and cell lines. Bulk global proteomics and flow cytometry-based studies are planned to confirm our genetic discoveries. Summary: Our rigorous computational and experimental approach provides valuable insights into tumor-specific cell surface proteins, which hold promise as potential targets for innovative and precision immunotherapeutic interventions in the realm of AML treatment.

Construction of an acute myeloid leukemia prognostic model based on m6A-related efferocytosis-related genes.

One of the most prevalent hematological system cancers is acute myeloid leukemia (AML). Efferocytosis-related genes (ERGs) and N6-methyladenosine (m6A) have an important significance in the progression of cancer, and the metastasis of tumors. The AML-related data were collected from The Cancer Genome Atlas (TCGA; TCGA-AML) database and Gene Expression Omnibus (GEO; GSE9476, GSE71014, and GSE13159) database. The "limma" R package and Venn diagram were adopted to identify differentially expressed ERGs (DE-ERGs). The m6A related-DE-ERGs were obtained by Spearman analysis. Subsequently, univariate Cox and Least Absolute Shrinkage and Selection Operator (LASSO) were used to construct an m6A related-ERGs risk signature for AML patients. The possibility of immunotherapy for AML was explored. The pRRophetic package was adopted to calculate the IC50 of drugs for the treatment of AML. Finally, the expression of characterized genes was validated by quantitative reverse transcription-PCR (qRT-PCR). Based on m6A related-DE-ERGs, a prognostic model with four characteristic genes (UCP2, DOCK1, SLC14A1, and SLC25A1) was constructed. The risk score of model was significantly associated with the immune microenvironment of AML, with four immune cell types, 14 immune checkpoints, 20 HLA family genes and, immunophenoscore (IPS) all showing differences between the high- and low-risk groups. A total of 56 drugs were predicted to differ between the two groups, of which Erlotinib, Dasatinib, BI.2536, and bortezomib have been reported to be associated with AML treatment. The qRT-PCR results showed that the expression trends of DOCK1, SLC14A1 and SLC25A1 were consistent with the bioinformatics analysis. In summary, 4 m6A related- ERGs were identified and the corresponding prognostic model was constructed for AML patients. This prognostic model effectively stratified the risk of AML patients.

Sting Activation Improves T-Cell Engaging Immunotherapy of Acute Myeloid Leukemia

Background: Despite emerging therapeutic options for Acute myeloid leukemia (AML) patients, for the majority of relapsed/refractory patients the outcome remains poor. Allogeneic stem cell transplantation has proven the power of T cells in eradicating residual leukemic cells, but alternative strategies based on T-cell recruiting bispecifics have so far failed to induce sustained responses. Resistance is mediated by the immunosuppressive tumor-microenvironment and secretion of immune dampening metabolites by AML cells. We hypothesized that combining a CD33-directed T cell engager (CD33 BiTE ® molecule or AMG 330) with a cGAS-STING agonist has the potential to augment anti-leukemic activity. Methods: In vitro co-culture assays of human T cells and AML cell lines for up to 72 h were performed to study the effect of AMG 330 in combination with the physiological STING-agonist 2‘-3‘ cGAMP (cGAMP). The cytotoxicity against the AML cell lines HL-60, MOLM-13 and OCI-AML-3 was assessed by flow cytometry. T-cell proliferation, T-cell degranulation (CD107a), as well as Granzyme B and TRAIL expression were characterized by flow cytometry. The secretion of effector cytokines (IFNy, TNF, IL-2, IFNa2a, CXCL10) was analyzed via CBA and ELISA assays. To better understand how AMG 330 and cGAMP work together, immunoblotting, transcriptomic analysis (bulk RNA-sequencing) and comprehensive CRISPRCas9 knockout-studies were carried out. Results: We observed a significant increase in AMG 330-mediated cytotoxicity of AML target cells upon co-application of cGAMP (Fig1A). The latter observation was enhanced upon prolongation of the culture and was also notable at low effector-to-target ratios. Monotherapy or addition of cGAMP by itself exerted only minor cytotoxicity against AML cells. The increase in AMG 330 mediated cytotoxicity through cGAMP was accompanied by an increase in T-cell proliferation, degranulation, Granzyme B secretion and TRAIL expression. Moreover, we observed a strong increase in secretion of proinflammatory cytokines (IFNy, IL-2 and IFNa2a). Bulk RNA-sequencing of AMG 330 and cGAMP stimulated co-cultures revealed type-I-IFN signature in the target (HL-60) and the effector cell (T cells) cell population. In addition, distinct phenotypes were found in each cell population: T cells demonstrated a marked increase in TNFa, IFNy and GZMB expression through the co-treatment, whereas HL-60 cells displayed an IFNy signaling signature (Fig1B). Importantly, the responsiveness of AML target cells to STING activation in terms of interferon stimulated gene (ISG)-induction was increased by the co-treatment, evidenced by further upregulation of STAT1, IRF7, WARS, CXCL11, IFI16, RSAD2 and CXCL10. An increase in type-I-IFN production and subsequent ISG-induction could also be confirmed on the protein level by measuring IFNα2a and CXCL-10. Knockout-studies revealed that the cGAMP enhanced AMG 330-mediated cytotoxicity was fully dependent on target cell intrinsic STING and IRF3 signaling, but also dependent on functional IFNy and TNFa signaling. Notably, the increased IFNy production by T cells upon cGAMP addition was dependent on functional target cell STING and IFNy signaling. These observations imply a crosstalk between effector and target cells and an intricate interdependence between target cell intrinsic IFNγ- and STING-signaling. Of note, the deletion of the IFNy-receptor in AML target cells also reduced AMG 330-dependent cytotoxicity under steady state conditions. Finally, we demonstrated that the combination of IFNy, TNFa and cGAMP sufficed to induce an apoptotic cell death of AML cells, similar to the cGAMP containing T-cell-HL-60 cocultures. Conclusion: We propose a novel mechanism by which AMG 330-activated T cells prime and sensitize AML target cells in a forward feedback loop towards STING activation, leading to increased type-I-IFN production and induction of ISGs. The beneficial effect of physiological cGAMP in enhancing AMG 330-mediated cytotoxicity was accompanied by the pronounced expression of effector cytokines and an overall cytotoxic T-cell phenotype. We established a key role for interferon gamma in AMG 330-mediated cytotoxicity of AML cells and in rendering AML cells responsive to STING agonism. We are currently conducting in vivoexperiments to further validate our findings.

Developing a novel SARS-CoV-2 risk index to predict the prognostic and therapeutic effects in acute myeloid leukemia

There is growing evidence of a strong association between SARS-CoV-2 and cancer prognosis and treatment outcome. However, there are no reliable SARS-CoV-2 assessment models to accurately predict prognostic and therapeutic effects in acute myeloid leukemia (AML). Here, differentially expressed genes associated with SARS-CoV-2 were detected, and multiple Cox regression methods were used to construct a SARS-CoV-2 risk index (SC2RI). Then, RT-qPCR was used to validate the gene expression levels in the AML samples. Finally, we explored how the SC2RI affected prognosis, immune infiltration, immunotherapy, and drug sensitivity in AML. We found that CYB5R3 and CLIP4 had been confirmed as hub genes in AML and were used to generate the SC2RI. The datasets indicated that the SC2RI had a superior predictive impact on the prognosis of AML. In addition, high expression of immune checkpoints and numerous immunological infiltrations were substantially correlated with a high SC2RI. However, it responded poorly to immune checkpoint blockade, which may be related to T-cell dysfunction, lack of effective antigens, and deficiency of synaptic capacity. Moreover, a high SC2RI was less susceptible to mTOR-related pathway medications but more sensitive to cell cycle suppressors. Therefore, categorization based on SC2RI could enhance the prognostic prediction of AML and help identify novel therapeutic approaches.

AMPK activation induces immunogenic cell death in AML

AbstractSurvival of patients with acute myeloid leukemia (AML) can be improved by allogeneic hematopoietic stem cell transplantation (allo-HSCT) because of the antileukemic activity of T and natural killer cells from the donor. However, the use of allo-HSCT is limited by donor availability, recipient age, and potential severe side effects. Similarly, the efficacy of immunotherapies directing autologous T cells against tumor cells, including T-cell recruiting antibodies, chimeric antigen receptor T-cell therapy, and immune checkpoint inhibitors are limited in AML because of multiple mechanisms of leukemia immune escape. This has prompted a search for novel immunostimulatory approaches. Here, we show that activation of adenosine 5′-monophosphate–activated protein kinase (AMPK), a master regulator of cellular energy balance, by the small molecule GSK621 induces calreticulin (CALR) membrane exposure in murine and human AML cells. When CALR is exposed on the cell surface, it serves as a damage-associated molecular pattern that stimulates immune responses. We found that GSK621-treated murine leukemia cells promote the activation and maturation of bone marrow–derived dendritic cells. Moreover, vaccination with GSK621-treated leukemia cells had a protective effect in syngeneic immunocompetent recipients bearing transplanted AMLs. This effect was lost in recipients depleted of CD4/CD8 T cells. Together, these results demonstrate that AMPK activation by GSK621 elicits traits of immunogenic cell death and promotes a robust immune response against leukemia. Pharmacologic AMPK activation thus represents a new potential target for improving the activity of immunotherapy in AML.

Immunotherapy in Acute Myeloid Leukemia: A Literature Review of Emerging Strategies.

In the last twenty years, we have witnessed a paradigm shift in the treatment and prognosis of acute myeloid leukemia (AML), thanks to the introduction of new efficient drugs or approaches to refine old therapies, such as Gemtuzumab Ozogamicin, CPX 3-5-1, hypomethylating agents, and Venetoclax, the optimization of conditioning regimens in allogeneic hematopoietic stem cell transplantation and the improvement of supportive care. However, the long-term survival of non-M3 and non-core binding factor-AML is still dismal. For this reason, the expectations for the recently developed immunotherapies, such as antibody-based therapy, checkpoint inhibitors, and chimeric antigen receptor strategies, successfully tested in other hematologic malignancies, were very high. The inherent characteristics of AML blasts hampered the development of these treatments, and the path of immunotherapy in AML has been bumpy. Herein, we provide a detailed review of potential antigenic targets, available data from pre-clinical and clinical trials, and future directions of immunotherapies in AML.

Development of a gene edited next-generation hematopoietic cell transplant to enable acute myeloid leukemia treatment by solving off-tumor toxicity

Immunotherapy of acute myeloid leukemia (AML) has been challenging because the lack of tumor-specific antigens results in "on-target, off-tumor" toxicity. To unlock the full potential of AML therapies, we used CRISPR-Cas9 to genetically ablate the myeloid protein CD33 from healthy donor hematopoietic stem and progenitor cells (HSPCs), creating tremtelectogene empogeditemcel (trem-cel). Trem-cel is a HSPC transplant product designed to provide a reconstituted hematopoietic compartment that is resistant to anti-CD33 drug cytotoxicity. Here, we describe preclinical studies and process development of clinical-scale manufacturing of trem-cel. Preclinical data showed proof-of-concept with loss of CD33 surface protein and no impact on myeloid cell differentiation or function. At clinical scale, trem-cel could be manufactured reproducibly, routinely achieving >70% CD33 editing with no effect on cell viability, differentiation, and function. Trem-cel pharmacology studies using mouse xenograft models showed long-term engraftment, multilineage differentiation, and persistence of gene editing. Toxicology assessment revealed no adverse findings, and no significant or reproducible off-target editing events. Importantly, CD33-knockout myeloid cells were resistant to the CD33-targeted agent gemtuzumab ozogamicin invitro and in vivo. These studies supported the initiation of the first-in-human, multicenter clinical trial evaluating the safety and efficacy of trem-cel in patients with AML (NCT04849910).

STAT5 promotes PD-L1 expression by facilitating histone lactylation to drive immunosuppression in acute myeloid leukemia

sImmunotherapy is a revolutionized therapeutic strategy for tumor treatment attributing to the rapid development of genomics and immunology, and immune checkpoint inhibitors have successfully achieved responses in numbers of tumor types, including hematopoietic malignancy. However, acute myeloid leukemia (AML) is a heterogeneous disease and there is still a lack of systematic demonstration to apply immunotherapy in AML based on PD-1/PD-L1 blockage. Thus, the identification of molecules that drive tumor immunosuppression and stratify patients according to the benefit from immune checkpoint inhibitors is urgently needed. Here, we reported that STAT5 was highly expressed in the AML cohort and activated the promoter of glycolytic genes to promote glycolysis in AML cells. As a result, the increased-lactate accumulation promoted E3BP nuclear translocation and facilitated histone lactylation, ultimately inducing PD-L1 transcription. Immune checkpoint inhibitor could block the interaction of PD-1/PD-L1 and reactive CD8+ T cells in the microenvironment when co-culture with STAT5 constitutively activated AML cells. Clinically, lactate accumulation in bone marrow was positively correlated with STAT5 as well as PD-L1 expression in newly diagnosed AML patients. Therefore, we have illustrated a STAT5-lactate-PD-L1 network in AML progression, which demonstrates that AML patients with STAT5 induced-exuberant glycolysis and lactate accumulation may be benefited from PD-1/PD-L-1-based immunotherapy.

Immune checkpoint molecule DNAM-1/CD112 axis is a novel target for natural killer-cell therapy in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a hematologic malignancy that frequently relapses, even if remission can be achieved with intensive chemotherapy. One known relapse mechanism is the escape of leukemic cells from immune surveillance. Currently, there is no effective immunotherapy for AML because of the lack of specific antigens. Here, we aimed to elucidate the association between CD155 and CD112 in AML cell lines and primary AML samples and determine the therapeutic response. Briefly, we generated NK-92 cell lines (NK-92) with modified DNAX-associated molecule 1 (DNAM-1) and T-cell immunoglobulin and ITIM domain (TIGIT), which are receptors of CD155 and CD112, respectively. Analysis of 200 cases of AML indicated that the survival of patients with high expression of CD112 was shorter than that of patients with low expression. NK-92 DNAM-1 exhibited enhanced cytotoxic activity against AML cell lines and primary cells derived from patients with AML. DNAM-1 induction in NK-92 cells enhanced the expression of cytotoxicity-related genes, thus overcoming the inhibitory activity of TIGIT. Between CD155 and CD112, CD112 is an especially important target for natural killer (NK)-cell therapy of AML. Using a xenograft model, we confirmed the enhanced antitumor effect of NK-92 DNAM-1 compared with that of NK-92 alone. We also discovered that CD112 (Nectin-2), an immune checkpoint molecule belonging to the Nectin/Nectin-like family, functions as a novel target of immunotherapy. In conclusion, modification of the DNAM-1/CD112 axis in NK cells may be an effective novel immunotherapy for AML. Furthermore, our findings suggest that the levels of expression of these molecules are potential prognostic markers in AML.

Epitope editing enables targeted immunotherapy of acute myeloid leukaemia.

Despite the considerable efficacy observed when targeting a dispensable lineage antigen, such as CD19 in B cell acute lymphoblastic leukaemia1,2, the broader applicability of adoptive immunotherapies is hampered by the absence of tumour-restricted antigens3-5. Acute myeloid leukaemia immunotherapies target genes expressed by haematopoietic stem/progenitor cells (HSPCs) or differentiated myeloid cells, resulting in intolerable on-target/off-tumour toxicity. Here we show that epitope engineering of donor HSPCs used for bone marrow transplantation endows haematopoietic lineages with selective resistance to chimeric antigen receptor (CAR) T cells or monoclonal antibodies, without affecting protein function or regulation. This strategy enables the targeting of genes that are essential for leukaemia survival regardless of shared expression on HSPCs, reducing the risk of tumour immune escape. By performing epitope mapping and library screenings, we identified amino acid changes that abrogate the binding of therapeutic monoclonal antibodies targeting FLT3, CD123 and KIT, and optimized a base-editing approach to introduce them into CD34+ HSPCs, which retain long-term engraftment and multilineage differentiation ability. After CAR T cell treatment, we confirmed resistance of epitope-edited haematopoiesis and concomitant eradication of patient-derived acute myeloid leukaemia xenografts. Furthermore, we show that multiplex epitope engineering of HSPCs is feasible and enables more effective immunotherapies against multiple targets without incurring overlapping off-tumour toxicities. We envision that this approach will provide opportunities to treat relapsed/refractory acute myeloid leukaemia and enable safer non-genotoxic conditioning.

Single-cell transcriptomic profiling reveals immune cell heterogeneity in acute myeloid leukaemia peripheral blood mononuclear cells after chemotherapy.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterised by the rapid clonal expansion of abnormally differentiated myeloid progenitor cells residing in a complex microenvironment. However, the immune cell types, status, and genome profile of the peripheral blood mononuclear cell (PBMC) microenvironment in AML patients after chemotherapy are poorly understood. In order to explore the immune microenvironment of AML patients after chemotherapy, we conducted this study for providing insights into precision medicine and immunotherapy of AML. In this study, we used single-cell RNA sequencing (scRNA-seq) to analyse the PBMC microenvironment from five AML patients treated with different chemotherapy regimens and six healthy donors. We compared the cell compositions in AML patients and healthy donors, and performed gene set enrichment analysis (GSEA), CellPhoneDB, and copy number variation (CNV) analysis. Using scRNA-seq technology, 91,772 high quality cells of 44,950 PBMCs from AML patients and 46,822 PBMCs from healthy donors were classified as 14 major cell clusters. Our study revealed the sub-cluster diversity of T cells, natural killer (NK) cells, monocytes, dendritic cells (DCs), and haematopoietic stem cell progenitors (HSC-Prog) in AML patients under chemotherapy. NK cells and monocyte-DCs showed significant changes in transcription factor expression and chromosome copy number variation (CNV). We also observed significant heterogeneity in CNV and intercellular interaction networks in HSC-Prog cells. Our results elucidated the PBMC single-cell landscape and provided insights into precision medicine and immunotherapy for treating AML.

HLA-B*44 and the Bw4-80T motif are associated with poor outcome of relapse-preventive immunotherapy in acute myeloid leukemia.

HLA-B alleles are associated with outcomes in various pathologies, including autoimmune diseases and malignancies. The encodedHLA-B proteins are pivotal in antigen presentation to cytotoxic T cells, and some variants containing a Bw4 motif also serve as ligands to the killer immunoglobulin-like receptors (KIR) 3DL1/S1 of NK cells. We investigated the potential impact of HLA-B genotypes on the efficacy of immunotherapy for relapse prevention in acute myeloid leukemia (AML). Seventy-eight non-transplanted AML patients receiving HDC/IL-2 in the post-consolidation phase were genotyped for HLA-B and KIR genes. HLA-B*44 heralded impaired LFS (leukemia-free survival) and overall survival (OS), but the negative association with outcome was not shared across alleles of the HLA-B44 supertype. Notably, HLA-B*44 is one of few HLA-B44 supertype alleles containing a Bw4 motif with a threonine at position 80, which typically results in weak binding to the inhibitory NK receptor, KIR3DL1. Accordingly, a strong interaction between KIR3DL1 and Bw4 was associated with superior LFS and OS (p = 0.014 and p = 0.027, respectively). KIR3DL1+ NK cells from 80T-Bw4 donors showed significantly lower degranulation responses and cytokine responses than NK cells from 80I-Bw4 donors, suggesting impaired KIR3DL1-mediated education in 80T-Bw4 subjects. We propose that presence of a strong KIR3DL1+-Bw4 interaction improves NK cell education and thus is advantageous in AML patients receiving HDC/IL-2 immunotherapy for relapse prevention.

Single-cell RNA-seq reveals a microenvironment and an exhaustion state of T/NK cells in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous blood cancer. Effective immunotherapies for AML are hindered by a lack of understanding of the tumor microenvironment (TME). Here, we retrieved published single-cell RNA sequencing data for 128,688 cells derived from 29 bone marrow aspirates, including 21 AML patients and eight healthy donors. We established a global tumor ecosystem including nine main cell types. Myeloid, T, and NK cells were further re-clustered and annotated. Developmental trajectory analysis indicated that exhausted CD8+ T cells might develop via tissue residual memory T cells (TRM) in the AML TME. Significantly higher expression levels of exhaustion molecules in AML TRM cells suggested that these cells were influenced by the TME and entered an exhausted state. Meanwhile, the upregulation of checkpoint molecules and downregulation of granzyme were also observed in AML NK cells, suggesting an exhaustion state. In conclusion, our comprehensive profiling of T/NK subpopulations provides deeper insights into the AML immunosuppressive ecosystem, which is critical for immunotherapies.

NKG2A gene variant predicts outcome of immunotherapy in AML and modulates the repertoire and function of NK cells

BackgroundThe natural killer (NK) complex (NKC) harbors multiple genes such as KLRC1 (encoding NKG2A) and KLRK1 (encoding NKG2D) that are central to regulation of NK cell function. We aimed at...

Good manufacturing practice production of CD34+ progenitor-derived NK cells for adoptive immunotherapy in acute myeloid leukemia.

Allogeneic natural killer (NK) cell-based immunotherapy is a promising, well-tolerated adjuvant therapeutic approach for acute myeloid leukemia (AML). For reproducible NK cell immunotherapy, a homogenous, pure and scalable NK cell product is preferred. Therefore, we developed a good manufacturing practice (GMP)-compliant, cytokine-based ex vivo manufacturing process for generating NK cells from CD34+ hematopoietic stem and progenitor cells (HSPC). This manufacturing process combines amongst others IL15 and IL12 and the aryl hydrocarbon receptor antagonist StemRegenin-1 (SR1) to generate a consistent and active NK cell product that fits the requirements for NK cell immunotherapy well. The cell culture protocol was first optimized to generate NK cells with required expansion and differentiation capacity in GMP-compliant closed system cell culture bags. In addition, phenotype, antitumor potency, proliferative and metabolic capacity were evaluated to characterize the HSPC-NK product. Subsequently, seven batches were manufactured for qualification of the process. All seven runs demonstrated consistent results for proliferation, differentiation and antitumor potency, and preliminary specifications for the investigational medicinal product for early clinical phase trials were set. This GMP-compliant manufacturing process for HSPC-NK cells (named RNK001 cells) is used to produce NK cell batches applied in the clinical trial 'Infusion of ex vivo-generated allogeneic natural killer cells in combination with subcutaneous IL2 in patients with acute myeloid leukemia' approved by the Dutch Ethics Committee (EudraCT 2019-001929-27).

Aggrephagy-related patterns in tumor microenvironment, prognosis, and immunotherapy for acute myeloid leukemia: a comprehensive single-cell RNA sequencing analysis.

Acute myeloid leukemia (AML) is a complex mixed entity composed of malignant tumor cells, immune cells and stromal cells, with intra-tumor and inter-tumor heterogeneity. Single-cell RNA sequencing enables a comprehensive study of the highly complex tumor microenvironment, which is conducive to exploring the evolutionary trajectory of tumor cells. Herein, we carried out comprehensive analyses of aggrephagy-related cell clusters based on single-cell sequencing for patients with acute myeloid leukemia. A total of 11 specific cell types (T, NK, CMP, Myeloid, GMP, MEP, Promono, Plasma, HSC, B, and Erythroid cells) using t-SNE dimension reduction analysis. Several aggrephagy-related genes were highly expressed in the 11 specific cell types. Using Monocle analysis and NMF clustering analysis, six aggrephagy-related CD8+ T clusters, six aggrephagy-related NK clusters, and six aggrephagy-related Mac clusters were identified. We also evaluated the ligand-receptor links and Cell-cell communication using CellChat package and CellChatDB database. Furthermore, the transcription factors (TFs) of aggrephagy-mediated cell clusters for AML were assessed through pySCENIC package. Prognostic analysis of the aggrephagy-related cell clusters based on R package revealed the differences in prognosis of aggrephagy-mediated cell clusters. Immunotherapy of the aggrephagy-related cell clusters was investigated using TIDE algorithm and public immunotherapy cohorts. Our study revealed the significance of aggrephagy-related patterns in tumor microenvironment, prognosis, and immunotherapy for AML.

A Direct Comparison, and Prioritisation, of the Immunotherapeutic Targets Expressed by Adult and Paediatric Acute Myeloid Leukaemia Cells: A Systematic Review and Meta-Analysis.

Acute myeloid leukaemia (AML) is characterized by impaired myeloid differentiation resulting in an accumulation of immature blasts in the bone marrow and peripheral blood. Although AML can occur at any age, the incidence peaks at age 65. The pathobiology of AML also varies with age with associated differences in incidence, as well as the frequency of cytogenetic change and somatic mutations. In addition, 5-year survival rates in paediatrics are 60-75% but fall to 5-15% in older AML patients. This systematic review aimed to determine whether the altered genes in AML affect the same molecular pathways, indifferent of patient age, and, therefore, whether patients could benefit from the repurposing drugs or the use of the same immunotherapeutic strategies across age boundaries to prevent relapse. Using a PICO framework and PRISMA-P checklist, relevant publications were identified using five literature databases and assessed against an inclusion criteria, leaving 36 articles, and 71 targets for therapy, for further analysis. QUADAS-2 was used to determine the risk of bias and perform a quality control step. We then priority-ranked the list of cancer antigens based on predefined and pre-weighted objective criteria as part of an analytical hierarchy process used for dealing with complex decisions. This organized the antigens according to their potential to act as targets for the immunotherapy of AML, a treatment that offers an opportunity to remove residual leukaemia cells at first remission and improve survival rates. It was found that 80% of the top 20 antigens identified in paediatric AML were also within the 20 highest scoring immunotherapy targets in adult AML. To analyse the relationships between the targets and their link to different molecular pathways, PANTHER and STRING analyses were performed on the 20 highest scoring immunotherapy targets for both adult and paediatric AML. There were many similarities in the PANTHER and STRING results, including the most prominent pathways being angiogenesis and inflammation mediated by chemokine and cytokine signalling pathways. The coincidence of targets suggests that the repurposing of immunotherapy drugs across age boundaries could benefit AML patients, especially when used in combination with conventional therapies. However, due to cost implications, we would recommend that efforts are focused on ways to target the highest scoring antigens, such as WT1, NRAS, IDH1 and TP53, although in the future other candidates may prove successful.

Analysis of prognostic biomarker models and immune microenvironment in acute myeloid leukemia by integrative bioinformatics.

Acute myeloid leukemia (AML) is a hematological cancer driven on by aberrant myeloid precursor cell proliferation and differentiation. A prognostic model was created in this study to direct therapeutic care. Differentially expressed genes (DEGs) were investigated using the RNA-seq data from the TCGA-LAML and GTEx. Weighted Gene Coexpression Network Analysis (WGCNA) examines the genes involved in cancer. Find the intersection genes and construct the PPI network to discover hub genes and remove prognosis-related genes. A nomogram was produced for predicting the prognosis of AML patients using the risk prognosis model that was constructed using COX and Lasso regression analysis. GO, KEGG, and ssGSEA analysis were used to look into its biological function. TIDE score predicts immunotherapy response. Differentially expressed gene analysis revealed 1004 genes, WGCNA analysis revealed 19,575 tumor-related genes, and 941 intersection genes in total. Twelve prognostic genes were found using the PPI network and prognostic analysis. To build a risk rating model, RPS3A and PSMA2 were examined using COX and Lasso regression analysis. The risk score was used to divide the patients into two groups, and Kaplan-Meier analysis indicated that the two groups had different overall survival rates. Univariate and multivariate COX studies demonstrated that risk score is an independent prognostic factor. According to the TIDE study, the immunotherapy response was better in the low-risk group than in the high-risk group. We eventually selected out two molecules to construct prediction models that might be used as biomarkers for predicting AML immunotherapy and prognosis.