Functional characterization of cooperating MGA mutations in RUNX1::RUNX1T1 acute myeloid leukemia.

MGA (Max-gene associated) is a dual-specificity transcription factor that negatively regulates MYC-target genes to inhibit proliferation and promote differentiation. Loss-of-function mutations in MGA have been commonly identified in several hematological neoplasms, including acute myeloid leukemia (AML) with RUNX1::RUNX1T1, however, very little is known about the impact of these MGA alterations on normal hematopoiesis or disease progression. We show that representative MGA mutations identified in patient samples abolish protein-protein interactions and transcriptional activity. Using a series of human and mouse model systems, including a newly developed conditional knock-out mouse strain, we demonstrate that loss of MGA results in upregulation of MYC and E2F targets, cell cycle genes, mTOR signaling, and oxidative phosphorylation in normal hematopoietic cells, leading to enhanced proliferation. The loss of MGA induces an open chromatin state at promoters of genes involved in cell cycle and proliferation. RUNX1::RUNX1T1 expression in Mga-deficient murine hematopoietic cells leads to a more aggressive AML with a significantly shortened latency. These data show that MGA regulates multiple pro-proliferative pathways in hematopoietic cells and cooperates with the RUNX1::RUNX1T1 fusion oncoprotein to enhance leukemogenesis.

Usefulness of KIT mutant transcript levels for monitoring measurable residual disease in t (8;21) acute myeloid leukemia.

In addition to RUNX1::RUNX1T1 transcript levels, measurable residual disease monitoring using KIT mutant (KITmut ) DNA level is reportedly predictive of relapse in t (8; 21) acute myeloid leukemia (AML). However, the usefulness of KITmut transcript levels remains unknown. A total of 202 bone marrow samples collected at diagnosis and during treatment from 52 t (8; 21) AML patients with KITmut (D816V/H/Y or N822K) were tested for KITmut transcript levels using digital polymerase chain reaction. The individual optimal cutoff values of KITmut were identified by performing receiver operating characteristics curve analysis for relapse at each of the following time points: at diagnosis, after achieving complete remission (CR), and after Course 1 and 2 consolidations. The cutoff values were used to divide the patients into the KITmut -high (KIT_H) group and the KITmut -low (KIT_L) group. The KIT_H patients showed significantly lower relapse-free survival (RFS) and overall survival (OS) rates than the KIT_L patients after Course 1 consolidation (p = 0.0040 and 0.021, respectively) and Course 2 consolidation (p = 0.018 and 0.011, respectively) but not at diagnosis and CR. The <3-log reduction in the RUNX1::RUNX1T1 transcript levels after Course 2 consolidation was an independent adverse prognostic factor for RFS and OS. After Course 2 consolidation, the KIT_H patients with >3-log reduction in the RUNX1::RUNX1T1 transcript levels (11/45; 24.4%) had similar RFS as that of patients with <3-log reduction in the RUNX1::RUNX1T1 transcript levels. The combination of KITmut and RUNX1::RUNX1T1 transcript levels after Course 2 consolidation may improve risk stratification in t (8; 21) AML patient with KIT mutation.

t(2;2;21;8)(p21;q37;q22;q22), a novel four-way complex translocation involving variant t(8;21) in case of acute myeloid leukemia : A case report and literature review.

Chromosomal translocation serves as a crucial diagnostic marker in the classification of acute myeloid leukemia. Among the most prevalent cytogenetic abnormalities is t(8;21)(q22;q22), typically associated with the FAB subtype AML-M2. On occasion, alternative forms of t(8;21) have been observed. This report presents a case of AML with RUNX1::RUNX1T1, wherein the karyotype revealed t(2;2;21;8)(p21;q37;q22;q22), representing the first instance of a variant t(8;21) involving both chromosomes 2. The combination of routine karyotype analysis and fluorescence in situ hybridization proves to be an effective method for identifying complex translocations of t(8;21).

AML with RUNX1::RUNX1T1 Cooperating two Mutations Relapsed Quickly after Achieving CR.

BACKGROUND: Acute myeloid leukemia (AML) with t(8;21)(q22;q22.1); RUNX1::RUNX1T1 has a relatively favorable prognosis with a high complete remission rate and long disease-free survival. METHODS AND RESULTS: Here we describe a patient who had AML with t(8;21)(q22;q22.1); RUNX1::RUNX1T1. Cooperating mutations including KRAS and ASXL1, and with other abnormal karyotype del(17) and with a myelomonocytic differentiation. CONCLUSIONS: The patient relapsed despite achieving a morphologic complete remission (CR).

Single-cell RNA sequencing of a new transgenic t(8;21) preleukemia mouse model reveals regulatory networks promoting leukemic transformation.

T(8;21)(q22;q22), which generates the AML1-ETO fusion oncoprotein, is a common chromosomal abnormality in acute myeloid leukemia (AML) patients. Despite having favorable prognosis, 40% of patients will relapse, highlighting the need for innovative models and application of the newest technologies to study t(8;21) leukemogenesis. Currently, available AML1-ETO mouse models have limited utility for studying the pre-leukemic stage because AML1-ETO produces mild hematopoietic phenotypes and no leukemic transformation. Conversely, overexpression of a truncated variant, AML1-ETO9a (AE9a), promotes fully penetrant leukemia and is too potent for studying pre-leukemic changes. To overcome these limitations, we devised a germline-transmitted Rosa26 locus AE9a knock-in mouse model that moderately overexpressed AE9a and developed leukemia with long latency and low penetrance. We observed pre-leukemic alterations in AE9a mice, including skewing of progenitors towards granulocyte/monocyte lineages and replating of stem and progenitor cells. Next, we performed single-cell RNA sequencing to identify specific cell populations that contribute to these pre-leukemic phenotypes. We discovered a subset of common myeloid progenitors that have heightened granulocyte/monocyte bias in AE9a mice. We also observed dysregulation of key hematopoietic transcription factor target gene networks, blocking cellular differentiation. Finally, we identified Sox4 activation as a potential contributor to stem cell self-renewal during the pre-leukemic stage.

KIT exon 17 mutations are predictive of inferior outcome in pediatric acute myeloid leukemia with RUNX1::RUNX1T1.

BACKGROUND: Pediatric core binding factor acute myeloid leukemia (CBF-AML), although considered a favorable risk subtype, exhibits variable outcomes primarily driven by additional genetic abnormalities, such as KIT mutations. PROCEDURE: In this study, we examined the prognostic impact of KIT mutations in 130 pediatric patients with CBF-AML, treated uniformly at a single center over 4 years (2017-2021). KIT mutations were detected via next-generation sequencing using a myeloid panel comprising 52 genes for most patients. RESULTS: Our findings revealed that KIT mutations were present in 31% of CBF-AML cases. Exon 17 KIT mutation was most commonly (72%) seen with notable occurrences at the D816 and N822 residue in 48% and 39% of cases, respectively. The 3-year cumulative incidence of relapse (CIR) and overall survival (OS) for patients with exon 17 KIT mutation were 36% and 40%, respectively, and was significantly worse in comparison to other site KIT mutations (3-year CIR: 11%; OS: 64%) and without KIT mutation (3-year CIR: 13%; OS:71%). Notably, the prognostic impact of KIT mutations was prominent in patients with RUNX1::RUNX1T1, but not in those with CBFB::MYH11 fusion. Additionally, a high KIT variant-allele frequency (VAF) (>33%) predicted for a higher disease relapse; 3-year CIR of 40% for VAF greater than 33% versus 7% for VAF less than 33%. When adjusted for site of KIT mutation and end-of-induction measurable residual disease, VAF greater than 33% correlated with poor OS (hazard ratio [HR]: 4.4 [95% CI: 1.2-17.2], p = .034). CONCLUSION: Exon 17 KIT mutations serve as an important predictor of relapse in RUNX1::RUNX1T1 pediatric AML. In addition, a high KIT VAF may predict poor outcomes in these patients. These results emphasize the need to incorporate KIT mutational analysis into risk stratification for pediatric CBF-AML.

Combination of eriocalyxin B and homoharringtonine exerts synergistic anti-tumor effects against t(8;21) AML.

Understanding the molecular pathogenesis of acute myeloid leukemia (AML) with well-defined genomic abnormalities has facilitated the development of targeted therapeutics. Patients with t(8;21) AML frequently harbor a fusion gene RUNX1-RUNX1T1 and KIT mutations as "secondary hit", making the disease one of the ideal models for exploring targeted treatment options in AML. In this study we investigated the combination therapy of agents targeting RUNX1-RUNX1T1 and KIT in the treatment of t(8;21) AML with KIT mutations. We showed that the combination of eriocalyxin B (EriB) and homoharringtonine (HHT) exerted synergistic therapeutic effects by dual inhibition of RUNX1-RUNX1T1 and KIT proteins in Kasumi-1 and SKNO-1 cells in vitro. In Kasumi-1 cells, the combination of EriB and HHT could perturb the RUNX1-RUNX1T1-responsible transcriptional network by destabilizing RUNX1-RUNX1T1 transcription factor complex (AETFC), forcing RUNX1-RUNX1T1 leaving from the chromatin, triggering cell cycle arrest and apoptosis. Meanwhile, EriB combined with HHT activated JNK signaling, resulting in the eventual degradation of RUNX1-RUNX1T1 by caspase-3. In addition, HHT and EriB inhibited NF-κB pathway through blocking p65 nuclear translocation in two different manners, to synergistically interfere with the transcription of KIT. In mice co-expressing RUNX1-RUNX1T1 and KITN822K, co-administration of EriB and HHT significantly prolonged survival of the mice by targeting CD34+CD38- leukemic cells. The synergistic effects of the two drugs were also observed in bone marrow mononuclear cells (BMMCs) of t(8;21) AML patients. Collectively, this study reveals the synergistic mechanism of the combination regimen of EriB and HHT in t(8;21) AML, providing new insight into optimizing targeted treatment of AML.

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.

[Clinical features and prognosis of core binding factor acute myeloid leukemia children in South China: a multicenter study].

Objective: To analyze the clinical features, efficacy and prognosis factors of core binding factor (CBF) acute myeloid leukemia (AML) children in South China. Methods: This was a retrospective cohort study. Clinical data of 584 AML patients from 9 hospitals between January 2015 to December 2020 was collected. According to fusion gene results, all patients were divided into two groups: CBF-AML group (189 cases) and non-CBF-AML group (395 cases). CBF-AML group were divided into AML1-ETO subgroup (154 cases) and CBFß-MYH11 subgroup (35 cases). Patients in CBF-AML group chosen different induction scheme were divided into group A (fludarabine, cytarabine, granulocyte colony stimulating factor and idarubicin (FLAG-IDA) scheme, 134 cases) and group B (daunorubicin, cytarabine and etoposide (DAE) scheme, 55 cases). Age, gender, response rate, recurrence rate, mortality, molecular genetic characteristics and other clinical data were compared between groups. Kaplan-Meier method was used for survival analysis and survival curve was drawn. Cox regression model was used to analyze prognostic factors. Results: A total of 584 AML children were diagnosed, including 346 males and 238 females. And a total of 189 children with CBF-AML were included, including 117 males and 72 females. The age of diagnosis was 7.3 (4.5,10.0)years, and the white blood cell count at initial diagnosis was 21.4 (9.7, 47.7)×109/L.The complete remission rate of the first course (CR1) of induction therapy, relapse rate, and mortality of children with CBF-AML were significantly different from those in the non-CBF-AML group (91.0% (172/189) vs. 78.0% (308/395); 10.1% (19/189) vs. 18.7% (74/395); 13.2% (25/189) vs. 25.6% (101/395), all P<0.05). In children with CBF-AML, the CBFß-MYH11 subgroup had higher initial white blood cells and lower proportion of extramedullary invasion than the AML1-ETO subgroup, with statistical significance (65.7% (23/35) vs. 14.9% (23/154), 2.9% (1/35) vs. 16.9% (26/154), both P<0.05). AML1-ETO subgroup had more additional chromosome abnormalities (75/154), especially sex chromosome loss (53/154). Compared with group B, group A had more additional chromosome abnormalities and a higher proportion of tumor reduction regimen, with statistical significance (50.0% (67/134) vs. 29.1% (16/55), 34.3% (46/134) vs. 18.2% (10/55), both P<0.05). Significant differences were found in 5-years event free survival (EFS) rate and 5-year overall survival (OS) rate between CBF-AML group and non-CBF-AML group ((77.0±6.4)%vs. (61.9±6.7)%,(83.7±9.0)%vs. (67.3±7.2)%, both P<0.05).EFS and OS rates of AML1-ETO subgroup and CBFß-MYH11 subgroup in children with CBF-AML were not significantly different (both P>0.05). Multivariate analysis showed in the AML1-ETO subgroup, CR1 rate and high white blood cell count (≥50×109/L) were independent risk factors for EFS (HR=0.24, 95%CI 0.07-0.85,HR=1.01, 95%CI 1.00-1.02, both P<0.05) and OS (HR=0.24, 95%CI 0.06-0.87; HR=1.01, 95%CI 1.00-1.02; both P<0.05). Conclusions: In CBF-AML, AML1-ETO is more common which has a higher extramedullary involvement and additional chromosome abnormalities, especially sex chromosome loss. The prognosis of AML1-ETO was similar to that of CBFß-MYH11. The selection of induction regimen group FLAG-IDA for high white blood cell count and additional chromosome abnormality can improve the prognosis.

Protein-Nanocaged Selenium Induces t(8;21) Leukemia Cell Differentiation via Epigenetic Regulation.

The success of arsenic in degrading PML-RARα oncoprotein illustrates the great anti-leukemia value of inorganics. Inspired by this, the therapeutic effect of inorganic selenium on t(8; 21) leukemia is studied, which has shown promising anti-cancer effects on solid tumors. A leukemia-targeting selenium nanomedicine is rationally built with bioengineered protein nanocage and is demonstrated to be an effective epigenetic drug for inducing the differentiation of t(8;21) leukemia. The selenium drug significantly induces the differentiation of t(8;21) leukemia cells into more mature myeloid cells. Mechanistic analysis shows that the selenium is metabolized into bioactive forms in cells, which drives the degradation of the AML1-ETO oncoprotein by inhibiting histone deacetylases activity, resulting in the regulation of AML1-ETO target genes. The regulation results in a significant increase in the expression levels of myeloid differentiation transcription factors PU.1 and C/EBPα, and a significant decrease in the expression level of C-KIT protein, a member of the type III receptor tyrosine kinase family. This study demonstrates that this protein-nanocaged selenium is a potential therapeutic drug against t(8;21) leukemia through epigenetic regulation.

Dual intron-targeted CRISPR-Cas9-mediated disruption of the AML RUNX1-RUNX1T1 fusion gene effectively inhibits proliferation and decreases tumor volume in vitro and in vivo.

Oncogenic fusion drivers are common in hematological cancers and are thus relevant targets of future CRISPR-Cas9-based treatment strategies. However, breakpoint-location variation in patients pose a challenge to traditional breakpoint-targeting CRISPR-Cas9-mediated disruption strategies. Here we present a new dual intron-targeting CRISPR-Cas9 treatment strategy, for targeting t(8;21) found in 5-10% of de novo acute myeloid leukemia (AML), which efficiently disrupts fusion genes without prior identification of breakpoint location. We show in vitro growth rate and proliferation reduction by 69 and 94% in AML t(8;21) Kasumi-1 cells, following dual intron-targeted disruption of RUNX1-RUNX1T1 compared to a non t(8;21) AML control. Furthermore, mice injected with RUNX1-RUNX1T1-disrupted Kasumi-1 cells had in vivo tumor growth reduction by 69 and 91% compared to controls. Demonstrating the feasibility of RUNX1-RUNX1T1 disruption, these findings were substantiated in isolated primary cells from a patient diagnosed with AML t(8;21). In conclusion, we demonstrate proof-of-principle of a dual intron-targeting CRISPR-Cas9 treatment strategy in AML t(8;21) without need for precise knowledge of the breakpoint location.

A Case of AML1-ETO Positive Acute Myeloid Leukemia Morphologically Similar to Chronic Myelogenous Leukemia.

BACKGROUND: The goal was to study the role of the morphology, immunophenotype, karyotype and fusion gene expression in a patient with diagnosis of AML1-ETO positive acute myeloid leukemia. METHODS: A case of AML1-ETO positive acute myeloid leukemia morphologically similar to chronic myelogenous leukemia was reported. The results of the morphology, immunophenotype, karyotype and fusion gene expression were analyzed by reviewing relevant literature. RESULTS: The patient was a young boy, at the age of 13, with clinical manifestations of intermittent fatigue and fever. Blood routine: White blood cell 142.6 x 109/L, Red blood cell 0.89 x 1012/L, Hemoglobin 41 g/L, Platelet 23 x 109/L, primitive cells account for 5%. Bone marrow smear: Granulocyte system hyperplasia is obvious, visible at each stage, primitive cells account for 17%, eosinophils, basophils, and phagocytic blood cells were observed. Flow cytometry showed myeloid primitive cell population was 4.14%, immature and mature granulocytes cell population was 85.22%, and eosinophil cell population was 0.61%. The results showed that the proportion of myeloid primitive cell was high, the expression of CD34 was enhanced, the expression of CD117 was partially absent, the expression of CD38 was weakened, the expression of CD19 was weak, and a few cells expressed CD56, and the phenotype was abnormal. The proportion of granulocyte series increased and the nucleus shifted to the left. The proportion of erythroid series was decreased, and the expression of CD71 was weakened. The results of fusion gene showed AML1-ETO positive. Karyotype analysis showed clonogenic abnormality t(8;21)(q22;q22). CONCLUSIONS: The peripheral blood and bone marrow images of patients with t(8;21)(q22;q22) AML1-ETO positive are the manifestations of chronic myelogenous leukemia, suggesting that cytogenetics and molecular genetics play an irreplaceable role in the diagnosis of acute myeloid leukemia, and the comprehensive diagnostic efficiency is significantly better than that of morphology.

A Case of Acute Mixed Cell Leukemia Resembling AML1-ETO Positive Acute Myeloid Leukemia.

BACKGROUND: The aim of the study was to improve the understanding of complex karyotype acute mixed cell leukemia containing pseudo Chediak-Higashi granules. METHODS: A case of acute mixed cell leukemia resembling AML1-ETO positive acute myeloid leukemia was reported. The results of morphological, immunophenotypic, and cytogenetic tests were analyzed by reviewing relevant literature. RESULTS: The patient was a young boy with clinical manifestations of recurrent fever. Bone marrow smear: Granulocyte system hyperplasia is obvious, visible at each stage, primitive cells account for 12%. These cells are large in volume, mostly round or class round, with abundant cell mass, stained gray blue, unbalanced development of some nuclear plasma, abnormal cytoplasmic staining, and visible "sunrise red" -like changes. Typical Auer bodies, pseudo Chadiak-Higashi granules and phagocytic erythroid substances were observed. The nuclei are irregular in shape, distorted and depressed, with fine chromatin and prominent large nucleoli. Bone marrow was extracted 3 days later, the bone marrow smear showed 65% primitive cells. The morphology of primitive cells was similar to that of 3 days ago. The results of flow cytometry showed that the primary/naive T cells in the samples possessed nuclear cells. Flow cytometry showed two groups of abnormal cells. One group accounted for 3.87% of nuclear cells and was a primitive/naive T-cell phenotype, mainly expressing: CD34+, CD7+, CD5+, CD2dim+, MPO-, CCD3 + part, CD3-, CD4-, CD8 -, CD117 -, CD13-, CD33-, HLA - DR -, CD10-, CD11b-, CD56-. The other group which accounted for 79.8% of the nuclear cells was monocyte phenotype, mainly expressing: CD34-, CD117-, CD13+ small amount, CD33+, HLA-DR-, CD11b+, CD14+, CD15+, CD36+, CD56+, CD64+, CD4+, CD85J+, CD85K + part. It matched the immunophenotype of acute mixed cell leukemia (T/MMPAL). Immunophenotypic leukemia-related fusion genes were negative, and karyotype analysis results were 45, XY, T (11; 12)(p13; Q13), -12-17, + mar [12]/90 < n > 4, idem x 2 [6]/46, XY. Combined with the above results, acute mixed cell leukemia was diagnosed. CONCLUSIONS: The flow cytometry is the gold standard in the diagnosis of acute mixed cell leukemia. The diagnosis of acute mixed cell leukemia requires the combination of clinical manifestations, cellular morphology, immunology, cytogenetics and molecular biology, and the comprehensive diagnosis efficiency is obviously better than that of morphology.

ABL1 kinase as a tumor suppressor in AML1-ETO and NUP98-PMX1 leukemias.

Deletion of ABL1 was detected in a cohort of hematologic malignancies carrying AML1-ETO and NUP98 fusion proteins. Abl1-/- murine hematopoietic cells transduced with AML1-ETO and NUP98-PMX1 gained proliferation advantage when compared to Abl1 + /+ counterparts. Conversely, overexpression and pharmacological stimulation of ABL1 kinase resulted in reduced proliferation. To pinpoint mechanisms facilitating the transformation of ABL1-deficient cells, Abl1 was knocked down in 32Dcl3-Abl1ko cells by CRISPR/Cas9 followed by the challenge of growth factor withdrawal. 32Dcl3-Abl1ko cells but not 32Dcl3-Abl1wt cells generated growth factor-independent clones. RNA-seq implicated PI3K signaling as one of the dominant mechanisms contributing to growth factor independence in 32Dcl3-Abl1ko cells. PI3K inhibitor buparlisib exerted selective activity against Lin-cKit+ NUP98-PMX1;Abl1-/- cells when compared to the Abl1 + /+ counterparts. Since the role of ABL1 in DNA damage response (DDR) is well established, we also tested the inhibitors of ATM (ATMi), ATR (ATRi) and DNA-PKcs (DNA-PKi). AML1-ETO;Abl1-/- and NUP98-PMX1;Abl1-/- cells were hypersensitive to DNA-PKi and ATRi, respectively, when compared to Abl1 + /+ counterparts. Moreover, ABL1 kinase inhibitor enhanced the sensitivity to PI3K, DNA-PKcs and ATR inhibitors. In conclusion, we showed that ABL1 kinase plays a tumor suppressor role in hematological malignancies induced by AML1-ETO and NUP98-PMX1 and modulates the response to PI3K and/or DDR inhibitors.