Immunotherapy Of Acute Myeloid Leukemia Research Articles

Lysosome-related genes predict acute myeloid leukemia prognosis and response to immunotherapy.

Acute myeloid leukemia (AML) is a highly aggressive and pathogenic hematologic malignancy with consistently high mortality. Lysosomes are organelles involved in cell growth and metabolism that fuse to form specialized Auer rods in AML, and their role in AML has not been elucidated. This study aimed to identify AML subtypes centered on lysosome-related genes and to construct a prognostic model to guide individualized treatment of AML. Gene expression data and clinical data from AML patients were downloaded from two high-throughput sequencing platforms. The 191 lysosomal signature genes were obtained from the database MsigDB. Lysosomal clusters were identified by unsupervised consensus clustering. The differences in molecular expression, biological processes, and the immune microenvironment among lysosomal clusters were subsequently analyzed. Based on the molecular expression differences between lysosomal clusters, lysosomal-related genes affecting AML prognosis were screened by univariate cox regression and multivariate cox regression analyses. Algorithms for LASSO regression analyses were employed to construct prognostic models. The risk factor distribution, KM survival curve, was applied to evaluate the survival distribution of the model. Time-dependent ROC curves, nomograms and calibration curves were used to evaluate the predictive performance of the prognostic models. TIDE scores and drug sensitivity analyses were used to explore the implication of the model for AML treatment. Our study identified two lysosomal clusters, cluster1 has longer survival time and stronger immune infiltration compared to cluster2. The differences in biological processes between the two lysosomal clusters are mainly manifested in the lysosomes, vesicles, immune cell function, and apoptosis. The prognostic model consisting of six prognosis-related genes was constructed. The prognostic model showed good predictive performance in all three data sets. Patients in the low-risk group survived significantly longer than those in the high-risk group and had higher immune infiltration and stronger response to immunotherapy. Patients in the high-risk group showed greater sensitivity to cytarabine, imatinib, and bortezomib, but lower sensitivity to ATRA compared to low -risk patients. Our prognostic model based on lysosome-related genes can effectively predict the prognosis of AML patients and provide reference evidence for individualized immunotherapy and pharmacological chemotherapy for AML.

Evolution of natural killer cell-targeted therapy for acute myeloid leukemia.

In hematologic oncology, acute myeloid leukemia (AML) presents a significant challenge due to its complex genetic landscape and resistance to conventional therapies. Despite advances in treatment, including intensive chemotherapy and hematopoietic stem cell transplantation (HSCT), the prognosis for many patients with AML remains poor. Recently, immunotherapy has emerged as a promising approach to improve outcomes by augmenting existing treatments. Natural killer (NK) cells, a subset of innate lymphoid cells, have garnered attention for their potent cytotoxic capabilities against AML cells. In this review, we discuss the role of NK cells in AML immunosurveillance, their dysregulation in patients with AML, and various therapeutic strategies leveraging NK cells in AML treatment. We explore the challenges and prospects associated with NK cell therapy, including approaches to enhance NK cell function, overcome immune evasion mechanisms, and optimize treatment efficacy. Finally, we emphasize the importance of further research to validate and refine patient-first NK cell-based immunotherapies for AML.

Induction of NK cell reactivity against acute myeloid leukemia by Fc-optimized CD276 (B7-H3) antibody.

Acute myeloid leukemia (AML) remains a therapeutic challenge despite recent therapeutic advances. Although monoclonal antibodies (mAbs) engaging natural killer (NK) cells via antibody-dependent cellular cytotoxicity (ADCC) hold promise in cancer therapy, almost none have received clinical approval for AML, so far. Recently, CD276 (B7-H3) has emerged as a promising target for AML immunotherapy, due to its high expression on leukemic blasts of AML patients. Here, we present the preclinical development of the Fc-optimized CD276 mAb 8H8_SDIE with enhanced CD16 affinity. We demonstrate that 8H8_SDIE specifically binds to CD276 on AML cell lines and primary AML cells and induces pronounced NK cell activation and degranulation as measured by CD69, CD25, and CD107a. Secretion of IFNγ, TNF, granzyme B, granulysin, and perforin, which mediate NK cell effector functions, was induced by 8H8_SDIE. A pronounced target cell-restricted lysis of AML cell lines and primary AML cells was observed in cytotoxicity assays using 8H8_SDIE. Finally, xenograft models with 8H8_SDIE did not cause off-target immune activation and effectively inhibited leukemia growth in vivo. We here present a novel attractive immunotherapeutic compound that potently induces anti-leukemic NK cell reactivity in vitro and in vivo as treatment option for AML.

Abstract NG05: Epitope editing enables targeted immunotherapy of acute myeloid leukemia

Abstract NG05: Epitope editing enables targeted immunotherapy of acute myeloid leukemia

Targeting PRAME for acute myeloid leukemia therapy.

Despite significant progress in targeted therapy for acute myeloid leukemia (AML), clinical outcomes are disappointing for elderly patients, patients with less fit disease characteristics, and patients with adverse disease risk characteristics. Over the past 10 years, adaptive T-cell immunotherapy has been recognized as a strategy for treating various malignant tumors. However, it has faced significant challenges in AML, primarily because myeloid blasts do not contain unique surface antigens. The preferentially expressed antigen in melanoma (PRAME), a cancer-testis antigen, is abnormally expressed in AML and does not exist in normal hematopoietic cells. Accumulating evidence has demonstrated that PRAME is a useful target for treating AML. This paper reviews the structure and function of PRAME, its effects on normal cells and AML blasts, its implications in prognosis and follow-up, and its use in antigen-specific immunotherapy for AML.

B Cell Maturation Antigen (BCMA) As a Novel and Promising Target for Immunotherapy for Acute Myeloid Leukemia

B Cell Maturation Antigen (BCMA) As a Novel and Promising Target for Immunotherapy for Acute Myeloid Leukemia

CLEC2A Is a Novel AML-Restricted Immunotherapeutic Target Enriched in KMT2A-Rearranged Acute Myeloid Leukemia

KMT2A rearrangements (KMT2A-r) are a frequent event in childhood acute myeloid leukemia (AML) and in treatment related AML in adults leading to a highly refractory and deadly subtype of AML regardless of age. Targeted therapies for KMT2A-r AML have had limited success with novel therapies urgently needed. Despite successes in other leukemias, immunotherapy in AML is in its infancy in part due to significant overlap of antigens expressed in AML and normal hematopoietic cells, which would limit efficacy and increase hematopoietic toxicity. We interrogated the AML transcriptome compared to normal hematopoiesis to identify optimal targets that are silent in normal hematopoieisis and are highly expressed in AML. Here we describe a novel immunotherapeutic target, CLEC2A, a member of the C-type lectin family, that is highly enriched in high-risk AML, particularly KMT2A-r AML and absent in normal hematopoeisis. We evaluated the association between KMT2A fusions and CLEC2A expression identifying CLEC2A as a putative oncoprotein and the potential for CLEC2A to serve as an immunotherapeutic target in high-risk AML via antibody-mediated cytotoxicity. Diagnostic specimens from 1,864 patients with AML (Pediatric 0-29 years from COG Phase III trials N=1486; Adult 18-88 years SWOG N=199 and TCGA-LAML N=179) were interrogated using contemporary NGS to identify genetic aberrations. Conventional karyotyping and mutational analysis were used to determine additional cytogenetic and molecular abnormalities. As CLEC2A was entirely absent in normal bone marrow (TPM 0-0.15), positive CLEC2A expression was defined as mRNA transcripts per million (TPM) >1. Although, CLEC2A was detected in only 18% of AML patients (252/1486; 17% pediatric and 90/378; 24% adult patients), it was highly enriched in those with KMT2A-r AML with 77% (N=193) of CLEC2A positive patients having KMT2A-r, accounting for 48% of the total KMT2A-r cohort, Fig 1A. CLEC2A expression was enriched in specific fusion subgroups: MLLT10 (80%), MLLT4 (71%), and MLLT3 (48%), Fig 1B. CLEC2A expression was low to absent in a majority of other patients, though was seen in other high-risk groups including non-KMT2A MLLT10 fusions, NUP98 fusions, CBFA2T3-GLIS2 fusions, and monosomy 7. CLEC2A is also expressed in a subtype of T-ALL (HOXA activated), which is a fusion driven T-ALL. We further evaluated a potential causal link between KMT2A fusions and CLEC2A expression and inquired whether KMT2A fusion proteins might directly bind to the CLEC2A promoter and induce CLEC2A expression. We used AutoCUT&RUN profiling of genome-wide occupancy of KMT2A oncoproteins to evaluate direct binding of the KMT2A fusion protein to the CLEC2A promoter in primary AML patient samples with three different KMT2A fusions (MLLT10, MLLT4 and MLLT3). We found direct binding of KMT2A fusion proteins to the CLEC2A promoter region, indicating a causal relationship between KMT2A fusions and CLEC2A expression. We then evaluated the potential of therapeutic targeting of CLEC2A by an antibody-drug conjugate using a HumZap cytotoxicy assay. CLEC2A antibody conjugated to saporin (a toxin with cytolytic activity when internalized) was incubated with CLEC2A+ and CLEC2A- cells with target-specific cytotoxicity evaluated by colorimetric assay. CLEC2A+ OCI-AML2 cells had significantly higher rate of cytotoxicity compared to CLEC2A- control cells at 10nM Ab-toxin conjugate. Clinical outcomes for patients with and without CLEC2A expression was evaluated. Patients with CLEC2A had significantly worse outcomes (EFS 23% vs. 45.5%, p<0.0001 and OS 38% vs. 63%, p<0.0001 for patients with and without CLEC2A expression, respectively). Given high rates of co-occurring KMT2A-r AML, particularly high-risk fusions (MLLT10 and MLLT4), we evaluated clinical outcomes within the KMT2A-r cohort as these patients have poor EFS and OS. KMT2A-r patients with CLEC2A had significantly worse EFS (24% vs. 39%, p=0.0063) and OS (38% vs. 60%, p=0.0002). Within the KMT2A-r cohort, high rates of relapse were seen for patients with CLEC2A expression compared to those without CLEC2A (72% vs. 55%, p=0.0039). Here we describe CLEC2A, a novel AML-restricted cell surface target that is an ideal immunotherapeutic target. CLEC2A is highly expressed in KMT2A-r AML, entirely absent in normal hematopoietic cells, directly and causally linked to the KMT2A fusion, and can be used for target-directed cytotoxicity.

4-1bb Enrichment Enhances Adoptive T Cell Therapy for Hematological Malignancies Using a Novel Approach for Ex Vivo Immune Cell Priming with DC/Tumor Fusion Vaccine

Introduction: While CAR T cells have transformed outcomes for patients with hematologic malignancies, application to patients with acute myeloid leukemia (AML) is limited by lack of surface antigens that differentiate between malignant and normal hematopoietic precursors. We have developed a personalized vaccine created by fusion of patient-derived tumor with autologous dendritic cells (DCs). The vaccine presents an array of tumor antigens stimulating polyclonal immunity. In a phase II clinical trial of patients with AML in first remission, vaccination with DC/AML fusions resulted in expansion of leukemia specific T cells associated with durable disease remission. The ex vivo generation of vaccine educated T cells provides a powerful substrate of effector cells for adoptive immunotherapy that are selective and capture tumor heterogeneity. This novel adoptive cell therapy enhances T-cell potency with potential to mediate disease regression. Enrichment of vaccine-educated T cells for activated antigen-specific effector cells via agonistic 4-1bb antibody-based selection further enhances for activation and memory cell phenotype; superior T cell efficacy was observed in AML and MM models. Methods: DC/tumor fusion vaccines were generated from C57BL/6J mice DCs and syngeneic C1498 mCh/luc + AML cells or C57BL/KaLwRij and syngeneic 5TGM1 mCh/luc + MM cells, respectively. T cells isolated from spleen were co-cultured with autologous irradiated DC/tumor fusions in presence of IL-2/7/15. Vaccine-educated T cells underwent selection with agonistic 4-1bb (3H3) followed by expansion with anti-CD3/CD28 activation beads. T cells were phenotyped for markers of activation (CD25/CD69), immune checkpoint (PD1/LAG3/TIM3), and memory (CD44+CD62L-) and for enrichment (anti-rat H&L). Cytotoxicity was evaluated on luminescent assay. Mice engrafted with C1498 mCh/luc + received the T cell therapy 7 days later. Disease progression and survival was monitored for 100 days by BLI. Results: In the C1498 model, vaccine-educated T cells demonstrated evidence of immune activation (CD25+CD69+) and memory (CD44+CD62L-) phenotype compared to unstimulated naïve T-cell controls (TN) (4-fold, p=0.1143; 1.3-fold, p=0.0195, respectively). Vaccine-educated T cells selected based on 4-1bb expression showed 9-fold higher expression of activation markers (p=0.0034) while memory markers were 4.4-fold higher compared to TN (p=0.0274). Selection enriched for 4-1bb + vaccine-educated T cells resulting in enhanced antigen-specific recognition as measured by induction of IFNg expression. Tumor specificity and activation was maintained following CD3/CD28-mediated expansion. The 4-1bb positive vaccine-educated T cells showed 11-fold enhanced cytotoxicity compared to TN at 10:1 E:T (P=0.0005), while vaccine-educated T cells showed 4-fold increase (P=0.0226). Phenotypic and functional analysis support 4 days as the optimal duration of time for T-cell vaccine education. Cell fold-expansion supports 3-5 days as the optimal duration for anti-CD3/CD28 expansion. In vivo, 60% of mice treated with 4-1bb + vaccine-educated cells were alive at 60 days vs 20% treated with unselected vaccine-educated cells. In the 5TGM1 model, activation, memory, immune checkpoint phenotype and cytotoxicity were increased in vaccine-educated T cells selected for 4-1bb expression. Conclusion: Vaccine-educated T cells represent a unique platform for adoptive immunotherapy for AML further enhanced by selection for 4-1bb expression by agonistic antibody. T-cell stimulation by fusion vaccine and subsequent enrichment by agonistic 4-1bb selection enhances cytotoxicity, activation and memory cell phenotype. Thus, ex vivo vaccine stimulation and 4-1bb selection presents a novel approach for cell-based immunotherapy for AML.

MP0533, a CD3-Engaging Darpin Targeting CD33, CD123, and CD70 in Patients with Relapsed/Refractory AML or MDS/AML: Preliminary Results of a Phase 1/2a Study

BACKGROUND: The development of targeted immunotherapy for acute myeloid leukemia (AML) is challenging due to the clonal heterogeneity of the disease and the lack of single AML-specific target antigens. MP0533 is a multi-specific, half-life extended CD3-engaging DARPin (Designed Ankyrin Repeat Protein). It simultaneously targets CD33, CD123, and CD70, thereby enabling avidity-driven T cell-mediated killing of leukemic stem cells (LSCs) and malignant blast cells known to co-express these targets. MP0533's affinity to each target is tuned to preferentially kill malignant cells which co-express at least two of these three antigens, while preserving a therapeutic window towards healthy cells. MP0533 was shown to induce a tumor-specific endogenous T-cell response in ex vivo samples from AML patients and led to T-cell-mediated eradication of tumors in AML xenograft mouse models, with limited cytokine release or other serious adverse reactions (Bianchi et al., Blood 2022;140[Suppl 1]:2251-2). Herein, we present early observations of the first three MP0533 dose-escalation regimens (DR) of the ongoing first-in-human, multicenter, single-arm, open-label, phase 1/2a study. METHODS: The objectives of this ongoing trial are to evaluate the safety/tolerability, pharmacokinetics (PK), and pharmacodynamics, as well as preliminary antileukemic activity of MP0533 monotherapy in patients with relapsed/refractory (R/R) AML or myelodysplastic syndrome (MDS)/AML (NCT05673057). Patients with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2, and a life expectancy of ≥12 weeks are eligible. Up to 45 patients will be enrolled in the dose-escalation part of the trial. Patients receive MP0533 once per week starting at day 15 after a stepwise dose increase on days 1, 5, and 8, and until disease progression or unacceptable toxicity. One cycle corresponds to 28 days. DR escalation of MP0533 is guided by two Bayesian logistic regression models estimating the joint probability of cytokine release syndrome (CRS) and non-CRS dose-limiting toxicities (DLTs). Bone marrow examinations are performed at weeks 4, 8, and 12. Responses are determined using 2022 European LeukemiaNet (ELN) criteria with an assessment of hematologic improvement. Treatment-emergent adverse events (TEAEs) are assessed according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0. RESULTS: As of data cut-off (20 July 2023), 5 patients received ≥5 infusions of MP0533 across 3 DRs preceded by step-up dosing (1 patient each at DR 1 and DR 2, respectively, and 3 patients at DR 3), including 2 women and 3 men. The median age at enrollment was 79 years (range 68-81). All patients had adverse risk mutations. Patients received a median number of 1 prior antileukemic therapy line (range 1-2) and on average 2 cycles of MP0533 treatment (range 1-3). To date, one patient with a TP53 mutation achieved a morphologic leukemia-free state after 8 weeks and complete remission with incomplete hematologic recovery (CRi) after 12 weeks of treatment with DR 3. No DLTs have been observed in any DR so far. TEAEs considered to be related to MP0533 were Grade 2 infusion-related reaction (2 patients), Grade 1 CRS (2 patients), and Grade 1 diarrhea and fatigue (1 patient). No Grade ≥3 drug-related TEAEs were reported. The reported TEAEs (Figure) reflected the patients' disease course. Preliminary PK results up to DR 3 confirmed that all patients were exposed to MP0533 and exposure levels were broadly aligned with PK/PD model predictions of MP0533 in serum. CONCLUSION: The results of this ongoing phase 1/2a study indicated an acceptable safety profile of MP0533 monotherapy in 5 patients up to DR 3 with weekly infusions. Preliminary response data are encouraging, with one response observed in 1 of 2 patients evaluable for response in DR 3 to date.

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.

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.

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.