Survival In Acute Myeloid Leukemia Patients Research Articles

DNA-PKcs-mediated transcriptional regulation of TOP2B drives chemoresistance in acute myeloid leukemia.

Anthracyclines, topoisomerase II enzyme poisons that cause DNA damage, are the mainstay of acute myeloid leukemia (AML) treatment. However, acquired resistance to anthracyclines leads to relapse, which currently lacks effective treatment and is the cause of poor survival in individuals with AML. Therefore, the identification of the mechanisms underlying anthracycline resistance remains an unmet clinical need. Here, using patient-derived primary cultures and clinically relevant cellular models that recapitulate acquired anthracycline resistance in AML, we have found that GCN5 (also known as KAT2A) mediates transcriptional upregulation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in AML relapse, independently of the DNA-damage response. We demonstrate that anthracyclines fail to induce DNA damage in resistant cells, owing to the loss of expression of their target enzyme, TOP2B; this was caused by DNA-PKcs directly binding to its promoter upstream region as a transcriptional repressor. Importantly, DNA-PKcs kinase activity inhibition re-sensitized AML relapse primary cultures and cells resistant to mitoxantrone, and abrogated their tumorigenic potential in a xenograft mouse model. Taken together, our findings identify a GCN5-DNA-PKcs-TOP2B transcriptional regulatory axis as the mechanism underlying anthracycline resistance, and demonstrate the therapeutic potential of DNA-PKcs inhibition to re-sensitize resistant AML relapse cells to anthracycline.

5-methylcytosine RNA modification regulators-based patterns and features of immune microenvironment in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a highly heterogeneous malignant disease of the blood cell. The current therapies for AML are unsatisfactory and the molecular mechanisms underlying AML are unclear. 5-methylcytosine (m5C) is an important posttranscriptional modification of mRNA, and is involved in the regulation of mRNA stability, translation, and other aspects of RNA metabolism. However, based on our knowledge of published literature, the role of the m5C regulators has not been explored in AML till date. In this study, we clarified the expression and gene variants of m5C regulators in AML and found that most m5C regulators were differentially expressed and correlated with disease prognosis. We also found that the methylation status of certain m5C regulators (e.g., DNMT3A, DNMT3B) affects the survival of AML patients. Two m5C modification subtypes, and high- and low-risk subgroups identified based on the expression of m5C regulators showed significant differences in the prognosis as well as immune cell infiltration. In addition, most of the m5C regulators were found to be correlated with miRNA expression in AML, as well as IC50 values of many drugs. The miRNA and GSVA analysis were used to identify the different miRNAs and KEGG or hallmark pathways between high- and low-risk subgroups. We also built a prognostic model based on m5C regulators, which was validated by two GSE databases. To verify the reliability of our analysis and conclusions, qPCR was used to identify the expressions of m5C regulators between normal and AML. In summary, we comprehensively explored the molecular characteristics of m5C regulators and built a prognostic model in AML. We proposed new mechanistic insights into the role of m5C in multiple databases and clinical data, which may pave novel ways for the development of therapeutic strategies.

Dysregulated immune and metabolic pathways are associated with poor survival in adult acute myeloid leukemia with CEBPA bZIP in-frame mutations

Acute myeloid leukemia (AML) with CEBPA bZIP in-frame mutations (CEBPAbZIP-inf) is classified within the favorable-risk group by the 2022 European LeukemiaNet (ELN-2022). However, heterogeneous clinical outcomes are still observed in these patients. In this study, we aimed to investigate the mutation profiles and transcriptomic patterns associated with poor outcomes in patients with CEBPAbZIP-inf. One hundred and thirteen CEBPAbZIP-inf patients were identified in a cohort of 887 AML patients homogeneously treated with intensive chemotherapy. Concurrent WT1 or DNMT3A mutations significantly predicted worse survival in AML patients with CEBPAbZIP-inf. RNA-sequencing analysis revealed an enrichment of interferon (IFN) signaling and metabolic pathways in those with a shorter event-free survival (EFS). CEBPAbZIP-inf patients with a shorter EFS had higher expression of IFN-stimulated genes (IRF2, IRF5, OAS2, and IFI35). Genes in mitochondrial complexes I (NDUFA12 and NDUFB6) and V (ATP5PB and ATP5IF1) were overexpressed and were associated with poorer survival, and the results were independently validated in the TARGET AML cohort. In conclusion, concurrent WT1 or DNMT3A mutations and a dysregulated immune and metabolic state were correlated with poor survival in patients with CEBPAbZIP-inf, and upfront allogeneic transplantation may be indicated for better long-term disease control.

Prognostic significance of ASXL1 mutations in acute myeloid leukemia: A systematic review and meta-analysis.

Although genetic mutations in additional sex-combs-like 1 (ASXL1) are prevalent in acute myeloid leukemia (AML), their exact impact on the AML prognosis remains uncertain. Hence, the present article was carried out to explore the prognostic importance of ASXL1 mutations in AML. We thoroughly searched electronic scientific databases to find eligible papers. Twenty-seven studies with an overall number of 8,953 participants were selected for the current systematic review. The hazard ratio (HR) and 95% confidence interval (CI) for overall survival (OS), event-free survival (EFS), and relapse-free survival (RFS) were extracted from all studies with multivariate or univariate analysis. Pooled HRs and p-values were also calculated as a part of our work. The pooled HR for OS in multivariable analysis indicated that ASXL1 significantly diminished survival in AML patients (pooled HR: 1.67; 95% CI: 1.342-2.091). ASXL1 mutations may confer a poor prognosis in AML. Hence, they may be regarded as potential prognostic factors. However, more detailed studies with different ASXL1 mutations are suggested to shed light on this issue.

STAT3 in acute myeloid leukemia facilitates natural killer cell-mediated surveillance.

Acute myeloid leukemia (AML) is a heterogenous disease characterized by the clonal expansion of myeloid progenitor cells. Despite recent advancements in the treatment of AML, relapse still remains a significant challenge, necessitating the development of innovative therapies to eliminate minimal residual disease. One promising approach to address these unmet clinical needs is natural killer (NK) cell immunotherapy. To implement such treatments effectively, it is vital to comprehend how AML cells escape the NK-cell surveillance. Signal transducer and activator of transcription 3 (STAT3), a component of the Janus kinase (JAK)-STAT signaling pathway, is well-known for its role in driving immune evasion in various cancer types. Nevertheless, the specific function of STAT3 in AML cell escape from NK cells has not been deeply investigated. In this study, we unravel a novel role of STAT3 in sensitizing AML cells to NK-cell surveillance. We demonstrate that STAT3-deficient AML cell lines are inefficiently eliminated by NK cells. Mechanistically, AML cells lacking STAT3 fail to form an immune synapse as efficiently as their wild-type counterparts due to significantly reduced surface expression of intercellular adhesion molecule 1 (ICAM-1). The impaired killing of STAT3-deficient cells can be rescued by ICAM-1 overexpression proving its central role in the observed phenotype. Importantly, analysis of our AML patient cohort revealed a positive correlation between ICAM1 and STAT3 expression suggesting a predominant role of STAT3 in ICAM-1 regulation in this disease. In line, high ICAM1 expression correlates with better survival of AML patients underscoring the translational relevance of our findings. Taken together, our data unveil a novel role of STAT3 in preventing AML cells from escaping NK-cell surveillance and highlight the STAT3/ICAM-1 axis as a potential biomarker for NK-cell therapies in AML.

C-Kit and Flt3 mutation status in acute myeloid leukaemia with cytogenetic correlation and prognosis: A series of 75 cases

Recent WHO classification (5 edition) has included Acute Myeloid Leukaemia (AML) with mutations in NPM1, CEBPA as separate entities along with AML with cytogenetic abnormalities in AML with recurrent genetic abnormalities. Even though C-kit and FLT3 mutations in AML have no diagnostic importance, prognostic significance in different subtypes of AML for the presence of these mutations are not the same.To assess the correlation between French American British (FAB) AML classification, specific cytogenetic abnormalities with C-kit, and FLT3 mutation in AML and to evaluate the prognosis and survival of AML patients with respect to cytogenetic abnormalities and C-kit and FLT3 mutation status. Retrospectively all AML cases in which C-kit and FLT3 mutation status was assessed were retrieved; C-kit D816V mutation status had been assessed by Real time PCR; For FLT3 mutation, both Internal tandem duplication (ITD) and D835V mutation status had been assessed using PCR and gel electrophoresis; The data regarding morphological with immunophenotypic diagnosis, conventional karyotyping, FISH for translocation 8;21 (t (8;21)) and inversion16 / translocation16;16 (t (16;16) were also retrieved in all these cases along with follow-up from hospital records. Total 75 cases were included; Male/ Female ratio was 1.21:1 (41/34); Median age was 31 (Range: 2 - 64); 18 cases had translocation 8;21 (t (8;21)); 3 cases showed inversion16 / translocation16;16 (t (16;16); Out of the 18 cases which showed t (8;21), 10 cases had associated loss of sex chromosome. Eight cases had C-kit D816V mutation; three of which had t (8;21) while two had inversion 16. 12 cases had FLT3 mutations among which nine were ITD while three had D835V mutation. On karyotyping, one of these cases showed hyperdiploidy while the majority had normal karyotype. A single case had both C-kit D816V mutation and FLT3 D835V mutation with inversion 16 on karyotyping; Most common type of AML in both cases with FLT3 mutation and C-kit mutation was AML-M2 (FAB); Commonest karyotyping abnormality for cases with C-kit mutation was t(8;21); while for FLT3 mutation, the majority had normal karyotype; The single case which had both C-kit D816V mutation and FLT3 D835V mutation was alive event-free at three-year follow-up. Both FLT3 ITD and TKD mutations had a worse prognosis in our study. However, AML cases with C-kit mutation had a similar prognosis comparable to C kit negative cases. A large-scale study is required to elucidate the significance of this.

Enhancing anti-AML activity of venetoclax by isoflavone ME-344 through suppression of OXPHOS and/or purine biosynthesis in vitro

Venetoclax (VEN), in combination with low dose cytarabine (AraC) or a hypomethylating agent, is FDA approved to treat acute myeloid leukemia (AML) in patients who are over the age of 75 or cannot tolerate standard chemotherapy. Despite high response rates to these therapies, most patients succumb to the disease due to relapse and/or drug resistance, providing an unmet clinical need for novel therapies to improve AML patient survival. ME-344 is a potent isoflavone with demonstrated inhibitory activity toward oxidative phosphorylation (OXPHOS) and clinical activity in solid tumors. Given that OXPHOS inhibition enhances VEN antileukemic activity against AML, we hypothesized that ME-344 could enhance the anti-AML activity of VEN. Here we report that ME-344 enhanced VEN to target AML cell lines and primary patient samples while sparing normal hematopoietic cells. Cooperative suppression of OXPHOS was detected in a subset of AML cell lines and primary patient samples. Metabolomics analysis revealed a significant reduction of purine biosynthesis metabolites by ME-344. Further, lometrexol, a purine biosynthesis inhibitor, synergistically enhanced VEN-induced apoptosis in AML cell lines. Interestingly, AML cells with acquired AraC resistance showed significantly increased purine biosynthesis metabolites and sensitivities to ME-344. Furthermore, synergy between ME-344 and VEN was preserved in these AraC-resistant AML cells. In vivo studies revealed significantly prolonged survival upon combination therapy of ME-344 and VEN in NSGS mice bearing parental or AraC-resistant MV4-11 leukemia compared to the vehicle control. This study demonstrates that ME-344 enhances VEN antileukemic activity against preclinical models of AML by suppressing OXPHOS and/or purine biosynthesis.

Targeting Epigenetic Resistance Mechanisms to PI3 Kinase Inhibition in Leukemic Stem Cells

Acute myeloid leukemia (AML) is a heterogeneous disease associated with poor survival and frequent relapse rates due to the inability to effectively target leukemic stem cells (LSCs) and acquired resistance to existing treatments. Development of treatments targeting LSCs and overcoming acquired resistance is crucial to improve the survival of AML patients. The PI3 kinase (PI3K)/AKT signaling pathway is activated in 80% of AML patients, across various AML subtypes, making PI3K an attractive therapeutic target for this disease. Several PI3K inhibitors, such as copanlisib, are clinically approved for other indications, with a reasonable toxicity profile. The effects of PI3K inhibitors on LSCs however, remains unknown. Several catalytic isoforms of PI3K are expressed in hematopoietic cells. To determine whether these PI3K isoforms play a role in LSC self-renewal, we performed colony assays on KMT2A-AF9 retrovirally transduced hematopoietic stem and progenitor cells (HSPCs) with either genetic deletion or pharmacologic inhibition of PI3K isoforms. We found that disruption of PI3K signaling reduces the self-renewal of LSCs and promotes myeloid differentiation in both mouse LSCs and human AML cell lines. KMT2A-AF9 induces leukemic transformation via aberrant upregulation of key self-renewal genes in hematopoietic progenitors, such as HoxA9 and Meis1 (PMID: 16862118). We found that pharmacologic inhibition of PI3K isoforms leads to a reduction in HoxA9 and Meis1 expression in LSC colonies. In the KMT2A-AF9 retroviral bone marrow transplantation model of AML, genetic deletion of the PI3K isoform Pik3ca leads to a reduction in LSC numbers in the bone marrow and prolonged survival (Fig 1A), although it is not curative. In addition, we observed significant prolongation of survival upon secondary transplantation of Pik3ca leukemic cells, suggesting a reduction in functional LSCs. Transcriptome analysis on LSCs isolated from pre-clinical bone marrow aspirates of KMT2A-AF9 primary transplant mice revealed downregulation of the target genes of HoxA9, an important driver of LSC self-renewal. We also observed enrichment of the targets of EZH2, a histone methyltransferase that catalyzes the H3K27 trimethylation repressive mark. AKT is known to phosphorylate EZH2 at Ser21, leading to inhibition of EZH2 function (PMID: 16224021). Therefore, we hypothesized that Pik3ca deletion can promote LSC differentiation by de-repressing EZH2 activation via AKT inactivation. To determine whether PI3K inactivation differentiates KMT2A-AF9 leukemic cells via dephosphorylation of EZH2, we performed Western blot analysis on bone marrow cells from Pik3ca KO and WT leukemic mice. As expected, Pik3ca deletion significantly reduced AKT phosphorylation. Unexpectedly, a reduction of total EZH2 protein levels was also observed in Pik3ca KO leukemic cells (Fig 1B), suggesting that EZH2 downregulation may be a non-genetic acquired resistance mechanism to PI3K inactivation. Histone proteomics analysis at two different timepoints after treatment with the PI3K inhibitor copanlisib revealed rapid and dynamic changes in histone modifications, including H3K27me3 and H3K4me3, consistent with epigenetic mechanisms of resistance to PI3K inhibition. EZH1, a homolog of EZH2, has been reported to compensate for EZH2 function (PMID: 26219303, PMID: 31189531). Thus, we hypothesize that PI3K inhibition may sensitize leukemic cells to EZH1/2 dual inhibition by reducing EZH2 levels. We found that dual inhibition with copanlisib and the EZH1/2 inhibitor valemetostat leads to a combinatorial effect to strongly decrease the proliferation of AML cell lines and AML patient samples. Additionally, we found that treatment of KMT2A-AF9 AML mice with these compounds induces myeloid differentiation and leads to a reduction in leukemia initiating cell capacity, as demonstrated by prolonged survival upon secondary transplantation. Overall, our data suggests that PI3K plays an important role in leukemic stem cells, and that EZH2 downregulation is a non-genetic mechanism of resistance to PI3K inhibition. Additionally, our findings indicate that combining PI3K inhibitors with EZH1/2 dual inhibitors could be a promising therapeutic approach to reduce LSCs, prevent resistance, and prolong survival in AML patients.

Impact of KMT2A-PTD Mutational Subgroups on Outcome of AML Patients after Induction Therapy and Allogeneic Hematopoietic Cell Transplantation

Introduction Genetic profiling of acute myeloid leukemia (AML) has increasingly revealed the possibility of adjusted therapeutic options. Recently, updates in genetic risk classifications introduced novel molecular features, but also excluded aberrations like partial tandem duplications in the lysine (K)-specific methyltransferase 2A ( KMT2A-PTD), formerly associated with adverse prognosis. Furthermore, there is growing evidence that molecular subtypes confer differential prognosis. Therefore, we analyzed a large cohort of AML pts to decipher the prognostic impact of KMT2A-PTD mutations ( KMT2A-PTD mut) including variants e9e3, e10e3 and e11e3 and their role in allogeneic hematopoietic cell transplantation (alloHCT). Methods Genomic DNA and total RNA of pts enrolled within the SAL registry (NCT03188874) was extracted at diagnosis. Samples were screened for KMT2A-PTD mut using the Mentype AMLplexQS Kit (Biotype, Dresden, Germany). Statistical as-treated analyses included standard statistical methods for categorial and continuous variables. Effects of alloHCT modeled as a time dependent covariate were estimated using Cox regression. Complete remission (CR) and survival rates were defined according to the current European Leukemia Net criteria. Logistic regression was used to evaluate CR rates and Cox regression to estimate hazard ratios (HR). The significance level was set at 0.05. All patients gave written informed consent. The study was conducted in accordance to the Declaration of Helsinki and was approved by the responsible ethics committees. Results Among 1409 pts, 206 pts harbored KMT2A-PTD mut, while 1203 pts showed wildtype (WT) alleles. KMT2A-PTD mut status was more often associated with secondary AML (p = .001) and less likely with concomitant NPM1 mutations (p < .001).Variants e9e3 (40.8%) and e10e3 (36.6%) were more frequently detected than e11e3 (22.6%). A total of 136 KMT2A-PTD mut and 859 WT pts received intensive induction therapy (IT). Rates of first complete remission (CR1) did not differ (p = .14). However, 5-year RFS was significantly lower in KMT2A-PTD mut pts compared to WT pts (15% vs. 25%), with median RFS of 23 vs. 29 months (HR = 1.4, p = .008). Accordingly, median OS was shorter (29 vs. 61 months; HR = 1.6, p = .002) and 5-year OS was lower for KMT2A-PTD mut pts compared to the WT counterparts (28% vs. 51%). In CR1, 81.6% of KMT2A-PTD mut pts proceeded to alloHCT and 18.4% of KMT2A-PTD mut pts received chemotherapy-based consolidation only. Median RFS improved from 15 to 37 months (HR = 0.5, p = .13) and chance of 5-year RFS enhanced from 17% to 21% when KMT2A-PTD mut pts underwent alloHCT in CR1 compared to KMT2A-PTD mut pts receiving chemo-consolidation.Median OS increased from 30 to 43 months when being allografted (HR = 0.5, p = .2), paralleled by increased rates of 5-year OS (33% chemo-consolidation vs. 47% alloHCT). Concerning KMT2A-PTD variants, there was no difference in likelihood to achieve CR1 between the three variants (p = .4). However, a trend towards shorter median OS ( e9e3: 41 months, e10e3:27 months, e11e3:27 months) and worse median RFS ( e9e3: 24 months, e10e3:23 months, e11e3:23 months) compared to WT pts (median OS: 61 months, median RFS: 29 months) could be revealed. Variant e10e3 was associated with worst prognosis (OS: HR = 2.1, p = .006; RFS: HR = 1.6, p = .06) and e9e3 with best prognosis (OS: HR = 1, p = .9; RFS: HR = 1.1, p = .7). e9e3 demonstrated best prognosis for both consolidation strategies. Median OS was not reached until last follow-up. Median RFS was higher in alloHCT pts (15 vs. 38 months, HR = 0.5, p = .3). e10e3 and e11e3 also had a higher median OS (12 vs. 43 months, HR = 0.3, p = .3 and 12 months vs. median survival not reached, HR = 0.2, p = .3, respectively) and RFS (11 vs. 41 months, HR = 0.3, p = .3 and 11 months vs. median survival not reached, HR = 0.2, p = .3, respectively) when allografted. Conclusions Based on our retrospective analysis, KMT2A-PTD mut are associated with reduced prognosis and survival in AML patients compared to WT pts. However, poor prognosis was effectively mitigated by alloHCT. Concerning KMT2A-PTD variants, we hypothesize alloHCT to be beneficial for improved survival. This effect seemed to be less pronounced for variant e9e3and more distinct for variant e11e3. Therefore, some KMT2A-PTD variants could be potentially implemented in AML risk stratification. However, prospective studies and larger patient numbers are needed.

Mutational Burden in High Risk Genes Do Not Affect Remission Rates and MRD Clearance in Elderly AML Patients Receiving CPX-351 Induction

Background: CPX-351 has recently been approved for the treatment of patients diagnosed with Acute Myeloid Leukemia (AML) arising from a previous myelodisplastic syndrome (s-AML) or secondary to chemotherapy (t-AML) as per former WHO 2016 classification. Minimal residual disease (MRD) assessment is a strong prognostic factor for relapse and shorter survival in AML patients undergoing intensive induction chemotherapy. Recent studies explored the role of genomic profile of AML influencing clinical outcome. In particular, Papaemmanuil et al showed that mutations in critical genes such as TP53, ASXL1, SRSF2 and RUNX1 are independently associated with a worse prognosis; moreover, co-occurrence of different high-risk mutations are predictors of dismal outcome when conventional chemotherapy is administered. Adverse risk mutations are particularly frequent in s-AML and t-AML. As clinical trials evaluating the prognostic relevance of MRD in the context of different molecular subsets mainly involve younger patients affected by de novo AML and receiving conventional 3+7 chemotherapy, there is a lack of data to define the impact of specific mutations on MRD clearance and prognosis in elderly s- AML and t-AML patients receiving CPX-351 induction. Aims: The aim of this study was to evaluate the prognostic impact of recurrent genic mutations and mutational burden at diagnosis on CR probability, MRD clearance and prognosis in a cohort of elderly s-AML or t-AML patients treated with CPX-351 induction chemotherapy. Methods: The study included 67 elderly patients (>60 year, median age 69, range 60-77) with a diagnosis of s-AML or t-AML according to WHO2016 classification, treated in our Center with CPX-351. Next generation sequencing (NGS) was performed using the Myeloid Solution panel by SOPHiA Genetics, encompassing 34 critical genes mutations. Sample have been processed on a Illumina MiSeq platform and the analysis performed with SOPHiA DDM® Software. MRD was analysed in all patients achieving complete remission (CR) with multicolour flow-cytometry, with a threshold of 0.1%. Results: AML patients were re-classified according to ELN 2022 risk score which was adverse, intermediate and favourable in 35, 29 and 3 patients, respectively. Median number of mutations for single patient (mutational burden) by NGS was 4 (range 2-9). Most frequent mutations involved: RUNX1 (44%), ASXL1 (37%), TP53 (33%), IDH1 (18%), IDH2 (14%), DNMT3A (14%), TET2 (8%). Four patients died before response assessment, mainly due to infections. Fifty-four patients (81%) achieved complete remission (CR) after first induction cycle; MRD evaluation was negative in 28/54 responding patients (52%). Univariate and multivariate analysis showed that factors traditionally associated with poor outcome such as ELN risk group, leukocytosis at diagnosis or previous treatment with hypomethylating agents did not affect the probability of CR rate and MRD negativity. Interestingly, having any high risk mutation, the combination of ≥2 high risk mutations or high mutational burden (having ≥ 4) did not affect CR probability and MRD negativity rate both in univariate and multivariate analysis. After cycle 2, the cumulative CR rate was 56/67 (84%), with 33/56 (59%) responding patients obtaining MFC MRD negativity. After a median follow up of 36 months (CI 95%; 22.4 -48.5 months), median OS was 19 months (CI 95% 13-24), whereas 2 year OS was 24%. Multivariate OS analysis revealed that negative MRD was the strongest independent predictor of longer survival (p<0.05). Notably, OS was not affected by mutational burden (29.2% and 27.6% for patients with less or more than 4 mutations, respectively, p=n.s, Fig.1). In landmark analysis, patients achieving CR and proceeding to allogeneic stem cell transplantation consolidation (HSCT) within 3 months from CR (N=13) had a significantly better outcome if compared to CR patients who did not receive HSCT or proceeded to transplant later (N= 41, 2-year OS: 84.6% and 31.7, respectively, p<0.03, Fig. 2). Conclusion : CPX-351 is able to induce good quality remission with high CR rate and MRD negativity, regardless of mutational burden, allowing a high number of elderly AML patients to undergo to HSCT. MRD evaluation has proven to be a strong prognostic tool also in the setting of elderly s-AML and t-AML patients receiving CPX-351. The prognostic value of high risk mutation seems to be less relevant in CPX-351 treated patients.

Casein Kinase II Inhibitor Sensitizes the Anti-Tumor Effect of EZH2 Inhibitor By Suppressing FOSL1 Expression in Acute Myeloid Leukemia

Background More effective combination therapy is emerging needed owing to the high mortality and poor outcome in acute myeloid leukemia (AML). The genomic amplification and overexpression of Enhancer of zeste homolog 2 (EZH2) occur in AML patients, and high expression of the Casein kinase 2 (CK2) is associated with poor survival in AML patients. Both EZH2 inhibitor and CK2 inhibitors are reported to inhibit cell proliferation and induce apoptosis in AML, however, the anti-leukemia effect of EZH2 inhibitor in combination with CK2 inhibitor has not been reported. The present study aims to investigate the effect of the combination of the EZH2 inhibitor DZNeP with CK2 inhibitor CX4945 in AML and possible underlying mechanisms. Methods A total of 40 newly diagnosed AML patients and 40 bone marrow control from healthy donors were obtained from our institute with the approval of the Ethics Committee. Cell Counting Kit-8 assay (CCK8), Cell cycle assay, and apoptosis assays were applied in THP-1 AML cells treated with control, DZNeP, CX4945, and CX4945+DZNeP. Global transcriptome analysis by RNA-seq was performed in U937 cells treated with 2μM DZNeP; 12.5μM CX4945 or vehicle for 48 hours. FOSL1 mRNA level was examined by qPCR; R2 genomics analysis and visualization application were used for comparison of the expression difference and the Kaplan-Meier overall survival analysis was conducted by the Gene Expression Profiling Interactive Analysis (GEPIA) platform. Results DZNeP only has little effect on cell proliferation arrest in THP-1 cells, but it significantly sensitized the effect of CX4945, and CalcuSyn analysis showed the synergistic effect of the CX4945+DZNeP (Fig.1A). The treatment of CX4945+DZNeP combination induced the G0/G1 phase arrest in THP-1 cells versus single-drug control (Fig. 1B). The combination also significantly increased the % of the apoptotic cells in THP-1 cells as well as the AML-5b primary cells from the patient compared to single-drug controls (p<0.0001) (Fig.1C). These data indicate the synergistic anti-tumor effect of CX4945+DZNep combination in AML. To understand the molecular mechanism underlying the synergy, the global transcriptome was identified in U937 cells. The significantly different expression genes (DEGs) were identified upon CX4945 treatment (∣log2FC∣>1.5, FDR<0.05), or DZNeP treatment (∣log2FC∣>1.0, FDR<0.05) versus vehicle-control; and 1908 overlapped DEGs are identified upon either drug treatment, in which 596 were intersected (429 genes changed in the same direction but 167 changed in the opposite (Fig. 1D). KEGG enrichment analyses showed the overlapped DEGs upon the drug treatment mainly involved in cell cycle, DNA replication, and MAPK signaling pathway. (Fig.1E). It is worth noting that FOSL1 is dramatically downregulated in U937 cells upon treatment of either DZNeP or CX4945 (Fig1.D); and qPCR data showed that the mRNA level of FOSL1 is significantly downregulated by CX4945+DZNeP, DZNeP or CX4945 compared to the vehicle control (p<0.0001) in THP-1 cells (Fig. 1F). These data indicated that the combination exerts its anti-tumor effect by down-regulation of FOSL1 expression in AML. FOSL1 is a key component of the AP-1 transcriptional complex, and overexpression of FOSL1 is associated with invasion, migration, proliferation, and stemness in human solid cancers. We also examined the FOSL1 mRNA level in 40 newly diagnosed patients with AML versus 40 normal bone marrow controls. Results showed the mRNA level of FOSL1 is significantly higher in AML patients than that of normal controls (p<0.001) (Fig1.G). Metadata analysis showed that FOSL1 high expression is associated with short overall survival (p=0.033) (Fig.1H). These results not only suggested the oncogenic role of FOSL1 in AML but also further supported the CX4945+DZNeP combination-induced anti-tumor effect mediated by suppressing FOSL1 expression in AML. The mechanism model is summarized in Fig. 1E. Conclusion The combination of EZH2 inhibitor DZNeP and CK2 inhibitor CX4945 exerts a potent synergistic effect on proliferation arrest, cell cycle arrest, and apoptosis in AML by suppressing FOSL1 expression. Our results reveal the oncogenic role of FOSL1 in AML. Our data also provide experimental hints for the potential clinical trial of the novel combination in AML patients.

ID2 Promotes Leukemogenesis and Chemoresistance of Acute Myeloid Leukemia By Modulating Cellular Oxidative Metabolism

Acute myeloid leukemia (AML) relies on quiescent leukemia stem cells (LSCs) to evade chemotherapy and drive disease relapse. Recent research indicates that a specific subset of LSCs, characterized by elevated oxidative phosphorylation and/or increased senescence, is responsible for chemotherapy resistance and relapse. While numerous genes associated with LSC maintenance and resistance have been identified, the underlying molecular mechanisms remain elusive. Inhibitor of DNA binding (ID) proteins, a group of Helix-Loop-Helix (HLH) transcription regulators, play a dominant role in regulating other HLH proteins (E proteins) required for the normal development of hematopoietic, muscle, neuronal, and other cells. Our previous investigation revealed that ID2 protects quiescent hematopoietic stem cells (HSCs) activation and exhaustion. Analysis of TCGA data sets demonstrated that high levels of ID2 expression are linked to reduced overall survival in AML patients, suggesting ID2 may be required to maintain some LSCs. Consistent with murine cell findings, we found that knocking down (KD) ID2 expression in human cord blood cells resulted in a loss of CD34+CD38- progenitors. Similarly, ID2-KD in AML cell lines (UT-7, MB-02, and M-07e) significantly reduced their leukemogenic potential in NSG mice, indicating that ID2 is essential for AML cell growth in vivo. Conversely, overexpressing ID2 in the human AML cell line M-07e enhanced their leukemic potential and burden in NSG mice, supporting the notion that ID2 may preserve LSC quiescence. Experiments with primary AML-Patient Derived Xenograft (AML-PDX) cells, which are more clinically relevant, also showed reduced leukemic potential in transplanted NSG mice upon ID2 knockdown. Additionally, ID2-KD cells displayed significantly lower levels of reactive oxygen species (ROS) and mitochondrial activity compared to control cells in both AML cell lines and PDX cells. As ROS-low cells are more susceptible to cytarabine (AraC), we investigated whether ID2-KD renders AML cells more susceptible to AraC. Indeed, MB-02-ID2-KD and UT7-ID2-KD cells showed increased sensitivity to AraC, while M-07e cells overexpressing ID2 exhibited resistance to AraC. Furthermore, AraC effectively eliminated ROS-low cells, but ROS-high cells remained resistant, supporting previous findings. To further investigate the effect of ID2-KD in AraC resistance in vivo, UT7-ID-KD and MB-02-ID2-KD transplanted NSG mice were treated with AraC and monitored for their overall survival, and it was observed that ID2-KD transplanted mice have significantly higher overall survival compared to the control cell transplanted mice. In conclusion, it can be said that ID2 plays a critical role in maintaining normal murine and human HSCs, and it likely contributes to leukemia development and chemotherapy resistance. Therefore, targeting ID2 may offer a promising therapeutic approach to selectively tackle LSCs, followed by chemotherapy and bone marrow transplantation to achieve complete remission.

Validation and Retraining of the Stellae-123 Gene Expression Signature Improved Risk Stratification in Taiwanese Acute Myeloid Leukemia Patients

The European Leukemia Net (ELN) recommendations provide valuable guidance in treatment decisions of patients with acute myeloid leukemia (AML). However, the genetic complexity and heterogeneity of AML are not fully covered, despite the critical role of gene expression analysis in risk stratification. Addressing this gap, the Stellae-123 score, an AI-based model that captures gene expression patterns, has demonstrated promising results in predicting survival of AML patients in four western cohorts. This study aims to assess the applicability of Stellae-123 in a Taiwanese cohort. Our cohort included 233 de novo AML patients, who were diagnosed and treated at the National Taiwan University Hospital and received standard induction therapy with 7+3 chemotherapy (or 5+2 in elder fit patients). All patients had sufficient bone marrow samples for DNA and RNA sequencing at diagnosis. The median age of the cohort was 41 years. Among the patients, ELN-2022 risk groups were distributed as follows: favorable (53.4%), intermediate (27.6%), and adverse (19%). Appling the original Stellae-123 model, which derived gene expression signatures from the BeatAML cohort RNA-seq data, our patients were categorized into low-, intermediate-, and high-risk groups, each representing 33% of the cohort. Overall, there were no differences in clinical features among these risk groups. The Stellae-123 risk groups partially aligned with the ELN risk groups, with more ELN low-risk patients in the Stellae-123 low-risk group and a higher proportion of ELN-high risk patients in the Stellae-123 high-risk group. Intriguingly, aside from mutations listed in ELN-2022, the Stellae-123 high-risk group had significantly less NRAS mutations than others. Cumulative hazards predicted by Stellae-123 model yielded a c-index of 0.612 for overall survival (OS), and the three risk groups had significantly different OS (p<0.001). We next postulated that since Stellae-123 relies on molecular features derived from the leukemic cells, it might serve as a biomarker of biological risk to predict relapse-free survival (RFS). The c-index calculated by Stellae-123 to predict RFS was 0.607, suggesting a reasonable ability to discriminate between different risk levels for RFS. Recognizing the potential variation between the original training cohort and the Taiwanese population, we addressed this concern by retraining the Stellae-123 model using Taiwanese RNAseq data. As a result, the model retraining led to significant improvements in the cross-validated c-indexes, with values of 0.656 (OS) and 0.636 (RFS), leading to an even more robust risk stratification (p<0.001, Figure 1A). As we noticed overlapping grouping between Stellae-123 and ELN-2022 in both pretrained and retrained models, we explored if Stellae-123's prognostic power was independent of ELN risk status by conducting a multivariable analysis. Remarkably, in both the pretrained and retrained Stellae-123 models, whether calculated as continuous values or categorized into three groups, they consistently demonstrated discriminative power in predicting OS and RFS, irrespective of age and ELN-2022 risk, suggesting that Stellae-123 could offer additional valuable information in predicting patient outcomes. Considering recent data showing that incorporating age helped improve the prognostication performance of ELN-2022 and that age was an independent risk factor for inferior outcome in our cohort, we examined how age could complement current risk stratification. Serial model testing revealed significant improvement of the prognostic models when age and transcriptomic data, especially the locally retrained Stellae-123 model, were combined with ELN-2022, evident by drastically declined Akaike Information Criterion (AIC) values. Time-dependent ROC curve analysis confirmed the potential of incorporating Stellae-123 and age to complement current risk stratification system (Figure 1B). In summary, we showed that the Stellae-123 predicted both OS and relapse risk in AML patients from Taiwan. Furthermore, we demonstrated that model retraining could improve prognostication by adapting the Stellae-123 to the particularities of Taiwanese patients. Given the remarkable extrapolation of the signature, it's suggested gene expression profile be incorporated in the risk stratification of AML patients who are candidates to intensive therapy.

Super-Enhancer-Driven IGF2BP2 and IGF2BP3 Promote DDX21 Expression in Acute Myeloid Leukemia

Background : Acute myeloid leukemia (AML) is a hematological malignancy with a median overall survival (OS) of 8.5 months and a 5-year OS rate of 24.0%. Considering that relapse and drug resistance are major obstacles affecting the survival of AML patients, it is urgent to investigate the deeper pathogenesis of AML. Epigenetic modifications are extensively involved in the leukemogenesis of AML, especially N6-methyladenosine (m 6A) RNA methylation and super-enhancers (SEs). However, the relationship between m 6A and SEs in AML has not been elucidated. Methods : Chromatin immunoprecipitation (ChIP) sequencing data from Gene Expression Omnibus (GEO) cohort were analyzed to search SE-related genes. The mechanisms of m 6A-binding proteins IGF2BP2 and IGF2BP3 on DDX21 were explored via methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP) and luciferase reporter assays. The potential roles of DDX21 in AML were verified through functional assays in vitro and in vivo. Results: Two SE-associated genes, IGF2BP2 and IGF2BP3, were identified in AML. There were high enrichment of H3K27ac, H3K4me1 and BRD4 in IGF2BP2 and IGF2BP3. BRD4 inhibitor JQ1 or BRD4 knockdown could lead to the suppression of IGF2BP2/3 expression, indicating the activation by SE machinery on the transcription of IGF2BP2/3. As m 6A-binding proteins, IGF2BP2 and IGF2BP3 enhanced the stability of DDX21 mRNA in an m 6A-dependent manner. DDX21 was highly expressed in AML patients, and elevated DDX21 expression suggested a poor survival. Functionally, DDX21 deficiency inhibited proliferation, facilitated apoptosis and arrested cell cycle of AML cells in vitro . I n vivo, knockdown of DDX21 substantially reduced tumor load and prolonged survival of recipient mice. Conclusions: Dysregulation of SE-IGF2BP2/IGF2BP3-DDX21 axis promotes the progression of AML, which paves the way to develop more effective therapeutic strategies.

The Impact of DNMT3A Mutation on Survival of AML Patients Receiving Allogeneic Hematopoietic Cell Transplantation in First Remission Depends on the Karyotype and Co-Occurring Mutations: On Behalf of the EBMT Acute Leukemia Working Party

DNMT3A gene mutations are more frequent in cytogenetically normal acute myeloid leukemia (AML) and frequently associated with NPM1 mutation and internal tandem duplication (ITD) of the FLT3 gene. However, DNMT3A mutation is not included as a distinct prognostic group in the recent European Leukemia Net (ELN) 2022 genetic risk classification of AML. In the context of allogeneic hematopoietic cell transplantation (allo-HCT), the prognostic value of DNMT3A is still not well studied. We evaluated, through the European Society for Blood and Marrow Transplantation (EBMT) registry, the impact of DNMT3A mutation, according to the karyotype, and NPM1 and FLT3-ITD mutation status, on post-transplant outcomes in AML patients receiving allo-HCT in first complete remission (CR1). We identified a total of 1374 adult AML patients (51% female, median age 57 years; range 18-78 years) with ELN-2022 intermediate- or poor-risk cytogenetics and available DNMT3A, FLT3ITD and NPM1 mutation status, who underwent their first allo-HCT in CR1 between 2013 and 2021. Of these, 631 (46%) patients had DNMT3A mutation. Patients with mutant DNMT3A were more likely to be female (57% versus 46%, p<0.001), to have normal cytogenetics (75% versus 54%, p<0.001), FLT3-ITD co-mutation (50% vs 23%, p<0.001), NPM1 co-mutation (60% vs 21%, p<0.001), and to receive a myeloablative conditioning and in vivo T cell depletion. After a median follow-up of 2.3 years, the 2-year leukemia free survival (LFS) and overall survival (OS) were 62% and 69%, respectively. The subsequent analysis was split according to baseline karyotype (normal or abnormal). Among 870 patients with normal cytogenetics, 472 (54%) patients had DNMT3A mutation. The 2-year relapse incidence (RI), LFS and OS were 24%, 63% and 70% respectively, in patients with DNMT3A mutation compared to 18%, 69% and 74% in patients with wild type DNMT3A. Among these patients with normal karyotype, multivariable analyses (MVA) were done within the 4 groups of FLT3-ITD and NPM1 mutations. Within the group of 324 patients with both FLT3-ITD and NPM1 positives, 244 were triple-positive (75%), LFS and OS were not significantly different with and without DNMT3A mutation (2y LFS: 62% vs 63%, HR: 0.98, p=0.95; 2y OS: 70% vs 73%, HR: 1.17, p=0.58). Conversely, among 146 patients with NPM1 mutation without FLT3-ITD, LFS was significantly lower for 91 patients with DNMT3A mutation (2y LFS: 68% vs 87%, HR: 2.70, p=0.02) but OS was not significantly different (2y OS: 79% vs 87%, HR: 2, p=0.13). On the other hand, among 318 with wild type NPM1 and without FLT3-ITD, LFS and OS were not significantly different for 108 patients with DNMT3A mutation compared to 210 patients with wild type DNMT3A (2y LFS: 62% vs 67%, HR: 1, p=0.98; 2y OS: 65% vs 71%, HR: 1.02, p=0.94). Conversely, among 82 patients with FLT3-ITD and wild type NPM1, 2-year LFS and OS were 53% and 58% respectively, for 29 patients with DNMT3A mutation compared to 71% and 76% respectively, for 53 patients with wild type DNMT3A. The number of patients didn't allow to perform MVA in this group. Finally, among 504 patients with abnormal cytogenetics, 159 (31%) patients had DNMT3A mutation. On MVA, DNMT3A mutation did not significantly affect LFS or OS. Poor-risk cytogenetics was associated with worse LFS, OS and increased RI. Older age negatively affected LFS and OS. In conclusion, in AML patients allografted in CR1, the impact of DNMT3A mutation depends on the karyotype and co-occurring mutations. Our data suggest that DNMT3A mutation negatively affects post-transplant survival selectively in patients with normal karyotype and either NPM1 mutation without FLT3-ITD, or patients with normal karyotype, FLT3-ITD and wild type NPM1. DNMT3A mutation shows no impact on post-transplant outcomes in other settings.

Higher PD-1/Tim-3 expression on IFN-γ+ T cells is associated with poor prognosis in patients with acute myeloid leukemia

ABSTRACT With the success of immune checkpoint inhibitors (ICI), such as anti- programmed death-1 (PD-1) antibody for solid tumors and lymphoma immunotherapy, a number of clinical trials with ICIs have been attempted for acute myeloid leukemia (AML) immunotherapy; however, limited clinical efficacy has been reported. This may be due to the heterogeneity of immune microenvironments and various degrees of T cell exhaustion in patients and may be involved in the IFN-γ pathway. In this study, we first characterized the percentage of PD-1+ and T cell immunoglobulin mucin-domain-containing-3 (Tim-3) +IFN-γ+ T cells in peripheral blood (PB) in AML compared with healthy individuals (HIs) by flow cytometry and further discussed the possibility of the reversal of T cell exhaustion to restore the secretion capacity of cytokines in T cells in AML based on blockade of PD-1 or Tim-3 (anti-PD-1 and anti-Tim-3 antibody) in vitro using a cytokine protein chip. A significantly increased percentage of PD-1+, Tim-3+, and PD-1+Tim-3+ IFN-γ+ T cells was observed in PB from patients with AML in comparison with HIs. Moreover, higher PD-1+IFN-γ+CD3+/CD8+ T cell levels were associated with poor overall survival in AML patients. Regarding leukemia cells, the percentage of Tim-3 in CD117+CD34+ AML cells was positively correlated with PD-1 in IFN-γ+CD4+ T cells. Furthermore, blocking PD-1 and Tim-3 may involve multiple cytokines and helper T cell subsets, mainly Th1 and Treg cells. Blockade of PD-1 or Tim-3 tends to restore cytokine secretion to a certain extent, a synergistic effect shown by the co-blockade of PD-1 and Tim-3. However, we also demonstrated the heterogeneity of secretory cytokines in ICI-treated T cells in AML patients.

How did the survival of acute myeloid leukemia change over the last ten years in our unit?

Acute myeloid leukemia (AML) is a hematological malignancy with high mortality rate. The treatment is especially challenging in patients older than 65 years, which is the large majority of those. For patients unfit for intensive chemotherapy regimens, only palliative cytoreduction and basic supportive care used to be the options in our unit. However, from 2018, the azacitidine-venetoclax combination has been a new therapeutic alternative. This treatment resulted in marked survival benefit in clinical trials, however, its impact on the daily clinical practice and the entire patient population is unclear. Our goal was to evaluate how the application of azacitidine-venetoclax changed the treatment and survival of AML patients in our practice. We retrospectively analyzed the available clinical data of all AML patients treated consecutively between January 1, 2011 and December 31, 2021 at the 3rd Department of Internal Medicine (from 2020 onward called Department of Internal Medicine and Hematology), examining their treatment depending on the time period of therapy (2011-2017 and 2018-2021). Patients with acute promyelocytic leukemia were excluded. 423 patients were diagnosed during this period. The number of cases showed a marked increase: in the first 7 years of our study, 184 patients were diagnosed, while this rose to 239 during the subsequent 4 years. The median age of patients was 67.6 years, with more than 60% of patients aged over 65. An improving trend can be observed in the overall survival: between 2011 and 2017, the median overall survival was 4.8 ± 0.9 months, while between 2018 and 2021, it was 8.3 ± 1.4 months (p = 0.051). Moreover, in the case of patients over 65 there was a significant overall survival improvement: 3.1 ± 0.5 vs. 4.9 ± 0.6 months (p = 0,01). The main factor behind this improvement could be that a large proportion of over 65 patients previously only fit for supportive care could now be treated with azacitidine-venetoclax: the percentage of actively treated patients grew from 57.1% to 75.3% in the second period. The survival of patients unfit for curative therapy and older than 65 showed a steady increase which can be attributed to the introduction of new therapeutic alternatives. Orv Hetil. 2023; 164(45): 1787-1794.

Single-cell RNA-seq reveals novel immune-associated biomarkers for predicting prognosis in AML patients with RUNX1::RUNX1T1

Acute myeloid leukemia (AML) with t(8;21)(q22;q22);(RUNX1::RUNX1T1) is highly heterogeneous and malignant. It has a relapse rate of nearly 40 %, resulting in clinical resistance or refractoriness to chemotherapy. Immune cells, particularly CD4(+) T and CD8(+) T lymphocytes, have been discovered to be dysfunctional in this condition, and functional recovery shows promising efficiency in preclinical trials. Here, with single-cell transcriptomic data from de novo AML patients with RUNX1::RUNX1T1 and at various stages following disease progression, we investigated the genes correlated with T-cell proliferation and activation. In leukemia cells, ADA, AHCY, GPN3 and LTBR were markedly highly expressed compared to those in T-cell at diagnosis, and they tended to increase with disease progression. Additionally, we discovered that AHCY was an effective biomarker to predict the overall survival as well as relapse-free survival of AML patients with RUNX1::RUNX1T1. The correlation of AHCY with infiltrated immune cells and immune checkpoints was also investigated. AML cohorts from two other independent studies, TCGA LAML (n = 145) and the GEO dataset (n = 104), also demonstrated an inferior outcome for AML patients with high AHCY expression. In conclusion, our research revealed that AHCY might function as a novel indicator to predict the prognosis and efficiency of T-cell proliferation and activation in AML patients with RUNX1::RUNX1T1.

The clinical features and prognostic implications of PTPN11 mutation in adult patients with acute myeloid leukemia in China.

The clinical significance of protein tyrosine phosphatase nonreceptor type 11 mutation (PTPN11mut ) in acute myeloid leukemia (AML) is underestimated. We collected the data of AML patients with mutated PTPN11 and wild-type PTPN11 (PTPN11wt ) treated at our hospital and analyzed their clinical characteristics and prognosis. Fifty-nine PTPN11mut and 124 PTPN11wt AML patients were included. PTPN11mut was more common in myelomonocytic and monocytic leukemia, and was more likely to co-mutate with KRAS, KMT2C, NRAS, U2AF1, NOTCH1, IKZF1, and USH2A mutations than PTPN11wt . The overall survival for AML patients with PTPN11mut was significantly shorter than that for those with PTPN11wt (p = 0.03). The negative impact of PTPN11mut on overall survival was pronounced in the "favorable" and "intermediate" groups of ELN2017 risk stratification, as well as in the wild-type NPM1 group (p = 0.01, p = 0.01, and p = 0.04). PTPN11mut is associated with distinct clinical and molecular characteristics, and adverse prognosis in AML patients.

A CD38/CD28xCD3 Trispecific T-Cell Engager (TCE) As a Potentially Active Agent for the Treatment of Older Patients with Acute Myeloid Leukemia (AML)

Background: Refractory disease is common and the outcome of elderly AML patients unable to receive intensive chemotherapy with acceptable side effects remains dismal. Thus, the identification of effective and safer drugs in AML is warranted. Immunotherapeutic strategies have shown extraordinary efficacy for the treatment of hematological malignancies, such as anti-CD38 monoclonal antibodies (mAbs) in multiple myeloma. Indeed, CD38 could represent a potential therapeutic target for AML too, not only because it is expressed in AML blasts, but also because AML patients with heterogeneous or low CD38 expression could benefit from T-cell based immunotherapies that are less dependent of the density of the targeted antigen. Aims: To evaluate CD38 as a potential therapeutic target in AML, and to determine the mode of action and preclinical efficacy of isatuximab (an IgG1 anti-CD38 mAb) and SAR442257, which is a new CD38/CD28xCD3 trispecific TCE. Results: We evaluated CD38 expression in bone marrow blasts from 241 newly diagnosed elderly AML patients by flow cytometry. Overall, 99.2% of AML patients expressed CD38, 55.6% with homogeneous pattern and 44.6% with heterogeneous pattern. CD38 expression had no impact in the overall survival of AML patients. Interestingly, CD38 expression increased 2-fold in MRD positive leukemic blasts compared with patient-matched blasts at diagnosis (n=10). At 24h and up to 96h, isatuximab (70 nM) decreased CD38 surface level in 3 AML cell lines expressing low (KG-1), intermediate (MOLM-13) and high or similar (OCI-AML3) basal CD38 density as compared to AML patients. Isatuximab (70 nM) did not induce complement-dependent cytotoxicity; by contrast, we observed a trend to an increased antibody-dependent cellular phagocytosis and significant antibody-dependent cellular cytotoxicity (ADCC) only in OCI-AML3 cells in the presence of purified NK cells. In 17 primary samples from AML patients with homogeneous vs heterogeneous CD38 expression, isatuximab (70 nM) showed a median of 19% and 4.5% tumor-cell death, respectively. Altogether, these findings indicated an association between CD38 density and the mode of action triggered by isatuximab and its efficacy. As such, AML patients with heterogeneous CD38 expression are less likely to respond to isatuximab, but might benefit from TCE that are less dependent of the density of the targeted antigen. Contrary to isatuximab, the CD38/CD28xCD3 TCE did not induce a dose- and time-dependent decrease in CD38 surface levels in any AML cell line measured with a non-competing anti-CD38 antibody, regardless of the CD38 antigen density. When we cultured PBMCs from healthy adults (n=6) with MOLM-13 cells (E:T ratio of 10:1) in the presence of isatuximab or the CD38/CD28xCD3 TCE (700 pM), we observed a significantly higher tumor cell lysis at 48h induced by the latter. The same effect of the CD38/CD28xCD3 TCE was observed with purified T-, but not NK-cells from healthy adults (n=3). To identify the immune cells involved in tumor cell lysis, we confirmed that isatuximab only induced NK-cell activation, whereas the CD38/CD28xCD3 TCE induced both T- and NK-cell activation, measured as percentage of CD69+ cells. Co-culture of purified NK- or T-cells with MOLM-13 cells in the presence of the CD38/CD28xCD3 TCE demonstrated a T-cell dependent bystander activation of NK cells. Furthermore, isatuximab downregulated CD38 in a dose-dependent manner, especially in NK cells, while the CD38/CD28xCD3 TCE did not. Surprisingly, the CD38/CD28xCD3 TCE induced CD38 upregulation on T cells, indicating again T-cell activation. The efficacy of isatuximab and the CD38/CD28xCD3 TCE was analyzed in primary AML samples (n=8) and blast lysis was observed in two and three samples, respectively. Notably, two of the three CD38/CD28xCD3 TCE-responders were resistant to isatuximab. CD38/CD28xCD3 TCE-mediated lysis was independent of CD38 expression and induced no significant off-target toxicity. Conclusions: CD38 is widely present in blasts from older AML patients but nearly half show heterogeneous expression. While isatuximab-driven ADCC in AML cell lines and primary samples is dependent on CD38 density in tumor cells, the CD38/CD28xCD3 TCE exerted its anti-tumor efficacy regardless of CD38 density. Thus, AML patients expressing both high and low/heterogenous levels of CD38 could benefit from T cell based immunotherapeutic strategies targeting CD38.