Internal Tandem Duplication Research Articles

Unveiling the signaling network of FLT3-ITD AML improves drug sensitivity prediction

Currently, the identification of patient-specific therapies in cancer is mainly informed by personalized genomic analysis. In the setting of acute myeloid leukemia (AML), patient-drug treatment matching fails in a subset of patients harboring atypical internal tandem duplications (ITDs) in the tyrosine kinase domain of the FLT3 gene. To address this unmet medical need, here we develop a systems-based strategy that integrates multiparametric analysis of crucial signaling pathways, and patient-specific genomic and transcriptomic data with a prior knowledge signaling network using a Boolean-based formalism. By this approach, we derive personalized predictive models describing the signaling landscape of AML FLT3-ITD positive cell lines and patients. These models enable us to derive mechanistic insight into drug resistance mechanisms and suggest novel opportunities for combinatorial treatments. Interestingly, our analysis reveals that the JNK kinase pathway plays a crucial role in the tyrosine kinase inhibitor response of FLT3-ITD cells through cell cycle regulation. Finally, our work shows that patient-specific logic models have the potential to inform precision medicine approaches.

Abstract 1966: The FLT3 tyrosine kinase receptor ITD mutation controls its expression and drug resistance in acute myeloid leukemia

Abstract In approximately 23% of AML patients, Fms-like tyrosine kinase 3 (FLT3) contains a gain-of-function, internal tandem duplication (ITD) mutation that is associated with an unfavourable prognosis. FLT3 is therefore seen as a promising therapeutic avenue for AML and as a result, a more complete understanding of its function and signalling may lead to additional targets and resistance mechanisms being identified. To date, several FLT3 inhibitors including Quizartinib (AC220), a second-generation ITD-selective tyrosine kinase inhibitor, have been developed, but single-agent clinical trials have not been overwhelmingly successful. In most preclinical studies, the inhibitory effects of FLT3 inhibitors are mainly evaluated using mutant-expressing models, however, most AML patients harbour both a wild-type and mutant FLT3 allele as well as presenting with high plasma levels of FLT3 ligand (FLT3L). We hypothesized that FLT3L could act through WT-FLT3 to influence the efficacy of FLT3 inhibitors in cells with heterozygous mutations. In this study, we have examined the role of FLT3 inhibition and FLT3L activation on the cellular localisation and downstream signalling in several AML cell lines, some of which express ITD mutations. We also looked at mechanisms by which FLT3L could impair the efficacy of FLT3 inhibitors. Our results revealed that the majority of FLT3 in the MV4-11 and MOLM-13 cells was intracellular. Inhibition of ITD-FLT3 with quizartinib led to a dramatic relocalisation of FLT3 to the cell surface. This effect was more pronounced in MV4-11 (FLT3ITD/ITD) than the heterozygous MOLM-13 (FLT3ITD/WT) cells that also express WT. This was accompanied by inhibition of ERK, AKT and STAT5 signalling pathways and resulted in cell death. Quizartinib induced cell death only in AML lines expressing FLT3-ITD mutations (MOLM-13 and MV4-11), and this could be antagonised by FLT3 ligand (FLT3L). The largest inhibitory effects were seen in heterozygous cells expressing a mutated and wild-type allele. FLT3 inhibition was associated with downregulation of the anti-apoptotic protein Mcl1 and upregulation of the pro-apoptotic BH3-only protein, Bim. Our experiments indicated that both of these proteins were regulated by both WT and ITD-FLT3 through the MAPK pathway. These results suggest that activation of FLT3 signalling by FLT3L confers resistance to quizartinib through upregulation of Mcl-1 and suppression of Bim expression. Taken together, our data suggest that FLT3 cell surface localisation and expression are controlled by ITD mutations and are key components of drug resistance. The data also suggests a novel therapeutic approach in patients with high plasma FLT3L levels, especially when using type II inhibitors such as quizartinib, is to co-target the MAPK/ERK pathway to abrogate the FLT3Lmediated resistance in FLT3-ITD AML. Citation Format: Taha Alqahtani, David MacEwan. The FLT3 tyrosine kinase receptor ITD mutation controls its expression and drug resistance in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1966.

Abstract 1844: Proteomic characterization of decitabine resistance in acute myeloid leukemia reveals signaling pathway crosstalk dampens the effectiveness of combination therapy

Abstract Acute myeloid leukemia (AML) is a deadly form of blood cancer primarily characterized by genetic abnormalities that guide treatment strategies. In recent years, several new therapies have emerged to target these genetic abnormalities such as gilteritinib or quizartinib, which target an internal tandem duplication (ITD) of the FLT3 gene. Additional drugs have been developed to target core cellular processes, such as venetoclax and the decitabine. Despite initial positive responses to these treatments, patients eventually relapse as they develop resistance to the individual drugs. One common approach to drug resistance is to add an additional drug to the treatment protocol. However, patients then develop further resistance to not just a single drug but multiple. As such, understanding the origins of both single and multi-drug resistance is crucial for improving treatment efficacy and clinical outcomes. Here, we present a collection of cell lines that have developed resistance to single, double, and triple drug combinations of gilteritinib, venetoclax, and decitabine. Through comprehensive global and phospho proteomics analyses under multiple conditions together with computational pathway interrogation, we unravel the underlying molecular mechanisms responsible for resistance. Leveraging our previous model of early and late drug resistance, we investigate the emergence of resistance to drug combinations involving gilteritinib alone, gilteritinib with venetoclax, gilteritinib with decitabine, and gilteritinib with venetoclax and decitabine. Through the characterization of molecular signatures for each drug combination, we identify molecular signals that are specific to combination treatments. Specifically, we find that the emergences of decitabine resistance alters the cellular pathways to effectively downregulate targets of venetoclax and gilteritinib, ultimately dampening the response to these drugs, regardless of co-administration. These findings provide a potential explanation for the limited success of combination treatments involving hypomethylating agents, as observed in recent clinical trials. Our results underscore the importance of monitoring proteome-level changes in single and combination drug treatments and shed light on the simple and intuitive reasons behind the lack of synergy often observed in combination therapies. In conclusion, we establish cell line models of resistance to various combinations of venetoclax, gilteritinib, and decitabine. By integrating global and phospo proteomic measurements, we gain a comprehensive understanding of the intricate molecular mechanisms underlying resistance. Citation Format: James C. Pino, Camilo Posso, Setareh Sharzehi, Sara Gosline, Chelsea Hutchinson-Bunch, Elie Traer, Paul D. Piehowski, Karin D. D. Rodland, Jeffrey W. Tyner, Tao Liu, Anupriya Agarwal. Proteomic characterization of decitabine resistance in acute myeloid leukemia reveals signaling pathway crosstalk dampens the effectiveness of combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1844.

Red blood cell membrane-coated FLT3 inhibitor nanoparticles to enhance FLT3-ITD acute myeloid leukemia treatment

FMS-like tyrosine kinase 3 (FLT3) is a viable and important therapeutic target for acute myeloid leukemia (AML). FLT3 internal tandem duplication (FLT3-ITD) mutations have been identified in approximately 30% of AML patients, and are associated with unfavorable prognosis, higher risk of relapse, drug resistance, and poor clinical outcome. Even FLT3 inhibitors have demonstrated promising efficacy, they cannot cure AML or even significantly extend the lives of patients with FLT3-ITD mutations. This is partly because of poor water solubility, insufficient membrane penetration and short half-life of small molecule inhibitors. Besides, the presence of enzymes like CYP3A4 in bone marrow accelerate the elimination and metabolism of FLT3 inhibitors, resulting in low plasma concentrations and side effects. Here we report the erythrocyte membrane-camouflaged FLT3 inhibitor nanoparticles to enhance FLT3-ITD AML treatment. Briefly, we physically coextruded red blood cell (RBC) membrane vesicles with nanoparticles derived from FLT3 inhibitor F30 to obtain F30@RBC-M, which exhibited comparable potent FLT3-ITD inhibitory effects compared to free F30in vitro, while displaying a higher potent antitumor efficacy in xenograft models due to the prolonged circulation properties. Furthermore, administration of F30@RBC-M significantly extended the survival of mice in a transplanted mouse model than F30 free drug. These findings suggest that RBC membrane-coated nanoparticles derived from FLT3 inhibitors hold promise as a tool to enhance the therapeutic efficacy to treat FLT3-ITD AML.

Gilteritinib as Post-Transplant Maintenance for AML With Internal Tandem Duplication Mutation of FLT3.

Allogeneic hematopoietic cell transplantation (HCT) improves outcomes for patients with AML harboring an internal tandem duplication mutation of FLT3 (FLT3-ITD) AML. These patients are routinely treated with a FLT3 inhibitor after HCT, but there is limited evidence to support this. Accordingly, we conducted a randomized trial of post-HCT maintenance with the FLT3 inhibitor gilteritinib (ClinicalTrials.gov identifier: NCT02997202) to determine if all such patients benefit or if detection of measurable residual disease (MRD) could identify those who might benefit. Adults with FLT3-ITD AML in first remission underwent HCT and were randomly assigned to placebo or 120 mg once daily gilteritinib for 24 months after HCT. The primary end point was relapse-free survival (RFS). Secondary end points included overall survival (OS) and the effect of MRD pre- and post-HCT on RFS and OS. Three hundred fifty-six participants were randomly assigned post-HCT to receive gilteritinib or placebo. Although RFS was higher in the gilteritinib arm, the difference was not statistically significant (hazard ratio [HR], 0.679 [95% CI, 0.459 to 1.005]; two-sided P = .0518). However, 50.5% of participants had MRD detectable pre- or post-HCT, and, in a prespecified subgroup analysis, gilteritinib was beneficial in this population (HR, 0.515 [95% CI, 0.316 to 0.838]; P = .0065). Those without detectable MRD showed no benefit (HR, 1.213 [95% CI, 0.616 to 2.387]; P = .575). Although the overall improvement in RFS was not statistically significant, RFS was higher for participants with detectable FLT3-ITD MRD pre- or post-HCT who received gilteritinib treatment. To our knowledge, these data are among the first to support the effectiveness of MRD-based post-HCT therapy.

Outcomes and prognosis of haploidentical haematopoietic stem cell transplantation in children with FLT3-ITD mutated acute myeloid leukaemia.

The presence of internal tandem duplication mutations in the FMS-like tyrosine kinase 3 receptor (FLT3-ITD) is a poor prognostic predictor in paediatric patients with acute myeloid leukaemia (AML). We evaluated the treatment outcomes and prognostic factors of 45 paediatric patients with FLT3-ITD AML who achieved complete remission before haploidentical haematopoietic stem cell transplantation (haplo-HSCT) at our institution from 2012 to 2021. Among the 45 patients, the overall survival (OS), event‑free survival (EFS), and cumulative incidence of relapse (CIR) rates were 74.9% ± 6.6%, 64.1% ± 7.2%, and 31.4% ± 7.1%, respectively, with 48.8 months of median follow-up. Univariate and multivariate analyses associated positive minimal residual disease (MRD) at pre-HSCT and non-remission (NR) after introduce 1 with inferior long-term survival. The 100-day cumulative incidence of grade II-IV acute graft-versus-host disease (aGVHD) was 35.6% ± 5.2%, and that of grade III-IV aGVHD was 15.6% ± 3.0% The overall 4-year cumulative incidence of chronic graft-versus-host disease after transplantation was 35.7% ± 9.8%, respectively. In conclusion, haplo-HSCT may be a feasible strategy for paediatric patients with FLT3-ITD AML, and pre-HSCT MRD status and NR after introduce 1 significantly affected the outcomes.

Leukemic mutation FLT3-ITD is retained in dendritic cells and disrupts their homeostasis leading to expanded Th17 frequency.

Dendritic cells (DC) are mediators between innate and adaptive immune responses to pathogens and tumors. DC development is determined by signaling through the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3) in bone marrow myeloid progenitors. Recently the naming conventions for DC phenotypes have been updated to distinguish between "Conventional" DCs (cDCs) and plasmacytoid DCs (pDCs). Activating mutations of FLT3, including Internal Tandem Duplication (FLT3-ITD), are associated with poor prognosis for acute myeloid leukemia (AML) patients. Having a shared myeloid lineage it can be difficult to distinguish bone fide DCs from AML tumor cells. To date, there is little information on the effects of FLT3-ITD in DC biology. To further elucidate this relationship we utilized CITE-seq technology in combination with flow cytometry and multiplex immunoassays to measure changes to DCs in human and mouse tissues. We examined the cDC phenotype and frequency in bone marrow aspirates from patients with AML to understand the changes to cDCs associated with FLT3-ITD. When compared to healthy donor (HD) we found that a subset of FLT3-ITD+ AML patient samples have overrepresented populations of cDCs and disrupted phenotypes. Using a mouse model of FLT3-ITD+ AML, we found that cDCs were increased in percentage and number compared to control wild-type (WT) mice. Single cell RNA-seq identified FLT3-ITD+ cDCs as skewed towards a cDC2 T-bet- phenotype, previously shown to promote Th17 T cells. We assessed the phenotypes of CD4+ T cells in the AML mice and found significant enrichment of both Treg and Th17 CD4+ T cells in the bone marrow and spleen compartments. Ex vivo stimulation of CD4+ T cells also showed increased Th17 phenotype in AML mice. Moreover, co-culture of AML mouse-derived DCs and naïve OT-II cells preferentially skewed T cells into a Th17 phenotype. Together, our data suggests that FLT3-ITD+ leukemia-associated cDCs polarize CD4+ T cells into Th17 subsets, a population that has been shown to be negatively associated with survival in solid tumor contexts. This illustrates the complex tumor microenvironment of AML and highlights the need for further investigation into the effects of FLT3-ITD mutations on DC phenotypes and their downstream effects on Th polarization.

Paclitaxel mediates the PI3K/AKT/mTOR pathway to reduce proliferation of FLT3‑ITD+ AML cells and promote apoptosis.

Acute myeloid leukemia (AML) with internal tandem duplication (ITD) mutations in the FLT3 tyrosine kinase tend to have a poor prognosis. FLT3-ITD can promote the progress of AML by activating the PI3K/AKT/mTOR pathway. Paclitaxel (PTX) is a natural anticancer drug that has been widely used in chemotherapy for multiple malignancies. The present study used the CCK-8 assay, flow cytometry, PCR and western blotting to explore the anti-leukemia effect and possible mechanisms of PTX on MV4-11 cells with the FLT3-ITD mutation and the underlying mechanism. As a result, it was found that PTX could inhibit proliferation of MV4-11 cells and promoted apoptosis by inhibiting the PI3K/AKT/mTOR pathway.

Postinduction molecular MRD identifies patients with NPM1 AML who benefit from allogeneic transplant in first remission

AbstractSelection of patients with NPM1-mutated acute myeloid leukemia (AML) for allogeneic transplant in first complete remission (CR1-allo) remains controversial because of a lack of robust data. Consequently, some centers consider baseline FLT3–internal tandem duplication (ITD) an indication for transplant, and others rely on measurable residual disease (MRD) status. Using prospective data from the United Kingdom National Cancer Research Institute AML17 and AML19 studies, we examined the impact of CR1-allo according to peripheral blood NPM1 MRD status measured by quantitative reverse transcription polymerase chain reaction after 2 courses of induction chemotherapy. Of 737 patients achieving remission, MRD was positive in 19%. CR1-allo was performed in 46% of MRD+ and 17% of MRD− patients. We observed significant heterogeneity of overall survival (OS) benefit from CR1-allo according to MRD status, with substantial OS advantage for MRD+ patients (3-year OS with CR1-allo vs without: 61% vs 24%; hazard ratio [HR], 0.39; 95% confidence interval [CI], 0.24-0.64; P < .001) but no benefit for MRD− patients (3-year OS with CR1-allo vs without: 79% vs 82%; HR, 0.82; 95% CI, 0.50-1.33; P = .4). Restricting analysis to patients with coexisting FLT3-ITD, again CR1-allo only improved OS for MRD+ patients (3-year OS, 45% vs 18%; compared with 83% vs 76% if MRD-); no interaction with FLT3 allelic ratio was observed. Postinduction molecular MRD reliably identifies those patients who benefit from allogeneic transplant in first remission. The AML17 and AML19 trials were registered at www.isrctn.com as #ISRCTN55675535 and #ISRCTN78449203, respectively.

Pim Kinase Inhibitors Increase Gilteritinib Cytotoxicity in FLT3-ITD Acute Myeloid Leukemia Through GSK-3β Activation and c-Myc and Mcl-1 Proteasomal Degradation.

FLT3-ITD is present in 25% of in AML, with continued poor outcomes. Combining Pim kinase inhibitors with the FDA-approved FLT3 inhibitor gilteritinib increases cytotoxicity in vitro and in vivo through activation of GSK-3β, which phosphorylates and posttranslationally downregulates c-Myc and Mcl-1. The data support efficacy of GSK-3βactivation in FLT3-ITD AML, and also support development of a clinical trial combining the Pim inhibitor TP-3654 with gilteritinib.

BRCC36 associates with FLT3-ITD to regulate its protein stability and intracellular signaling in acute myeloid leukemia.

Fms-like tyrosine kinase-3 (FLT3) is a commonly mutated gene in acute myeloid leukemia (AML). The two most common mutations are the internal-tandem duplication domain (ITD) mutation and the tyrosine kinase domain (TKD) mutation. FLT3-ITD and FLT3-TKD exhibit distinct protein stability, cellular localization, and intracellular signaling. To understand the underlying mechanisms, we performed proximity labeling with TurboID to identify proteins that regulate FLT3-ITD or -TKD differently. We found that BRCA1/BRCA2-containing complex subunit 36 (BRCC36), a specific K63-linked polyubiquitin deubiquitinase, was exclusively associated with ITD, not the wild type of FLT3 and TKD. Knockdown of BRCC36 resulted in decreased signal transducers and activators of transcription 5 phosphorylation and cell proliferation in ITD cells. Consistently, treatment with thiolutin, an inhibitor of BRCC36, specifically suppressed cell proliferation and induced cell apoptosis in ITD cells. Thiolutin efficiently affected leukemia cell lines expressing FLT3-ITD cell viability and exhibited mutual synergies with quizartinib, a standard clinical medicine for AML. Furthermore, mutation of the lysine at 609 of ITD led to significant suppression of K63 polyubiquitination and decreased its stability, suggesting that K609 is a critical site for K63 ubiquitination specifically recognized by BRCC36. These data indicate that BRCC36 is a specific regulator for FLT3-ITD, which may shed light on developing a novel therapeutic approach for AML.

CNS tumor with CREBBP::BCORL1 Fusion and pathogenic mutations in BCOR and CREBBP: expanding the spectrum of BCOR-altered tumors

The fifth edition of the World Health Organization (WHO) classification of central nervous system (CNS) tumors introduced the new tumor type CNS tumor with BCOR internal tandem duplication (ITD), characterized by a distinct DNA methylation profile and peculiar histopathological features, including a circumscribed growth pattern, ependymoma-like perivascular pseudorosettes, microcystic pattern, absent or focal GFAP immunostaining, OLIG2 positivity, and BCOR immunoreactivity. We describe a rare case of a CNS tumor in a 45-year-old man with histopathological and immunohistochemical features overlapping the CNS tumor with BCOR internal tandem duplication (ITD) but lacking BCOR immunostaining and BCOR ITD. Instead, the tumor showed CREBBP::BCORL1 fusion and pathogenic mutations in BCOR and CREBBP, along with a DNA methylation profile matching the “CNS tumor with EP300:BCOR(L1) fusion” methylation class. Two CNS tumors with fusions between CREBBP, or its paralog EP300, and BCORL1, and approximately twenty CNS tumors with CREBBP/EP300::BCOR fusions have been reported to date. They exhibited similar ependymoma-like features or a microcystic pattern, along with focal or absent GFAP immunostaining, and shared the same DNA methylation profile. Given their morphological and epigenetic similarities, circumscribed CNS tumors with EP300/CREBBP::BCOR(L1) fusions and CNS tumors with BCOR ITD may represent variants of the same tumor type. The ependymoma-like aspect coupled with the lack of diffuse GFAP immunostaining and the presence of OLIG2 positivity are useful clues for recognizing these tumors in histopathological practice. The diagnosis should be confirmed after testing for BCOR(L1) gene fusions and BCOR ITD.

FLT3 tyrosine kinase inhibition modulates PRC2 and promotes differentiation in acute myeloid leukemia.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 (FLT3-ITD) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3-ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML.

Feasibility and antitumour activity of the FGFR inhibitor erdafitnib in three paediatric CNS tumour patients.

Alterations of the fibroblast growth factor (FGF) signalling pathway are increasingly recognized as frequent oncogenic drivers of paediatric brain tumours. We report on three patients treated with the selective FGFR1-4 inhibitor erdafitinib. Two patients were diagnosed with a posterior fossa ependymoma group A (PFA EPN) and one with a low-grade glioma (LGG), harbouring FGFR3/FGFR1 overexpression and an FGFR1 internal tandem duplication (ITD), respectively. While both EPN patients did not respond to erdafitinib treatment, the FGFR1-ITD-harbouring tumour showed a significant decrease in tumour volume and contrast enhancement throughout treatment. The tumour remained stable 6months after treatment discontinuation.

Feasible diet and circadian interventions reduce invivo progression of FLT3-ITD-positive acute myeloid leukemia.

Acute myeloid leukemia (AML) with an internal tandem duplication in the fms-like tyrosine kinase receptor 3 gene (FLT3-ITD) is associated with poor survival, and few studies have examined the impact of modifiable behaviors, such as nutrient quality and timing, in this subset of acute leukemia. The influence of diet composition (low-sucrose and/or low-fat diets) and timing of diet were tested in tandem with anthracycline treatment in orthotopic xenograft mouse models. A pilot clinical study to test receptivity of pediatric leukemia patients to macronutrient matched foods was conducted. A role for the circadian protein, BMAL1 (brain and muscle ARNT-like 1), in effects of diet timing was studied by overexpression in FLT3-ITD-bearing AML cells. Reduced tumor burden in FLT3-ITD AML-bearing mice was observed with interventions utilizing low-sucrose and/or low-fat diets, or time-restricted feeding (TRF) compared to mice fed normal chow adlibitum. In a tasting study, macronutrient matched low-sucrose and low-fat meals were offered to pediatric acute leukemia patients who largely reported liking the meals. Expression of the circadian protein, BMAL1, was heightened with TRF and the low-sucrose diet. BMAL1 overexpression and treatment with a pharmacological inducer of BMAL1 was cytotoxic to FLT3-ITD AML cells. Mouse models for FLT3-ITD AML show that diet composition and timing slows progression of FLT3-ITD AML growth invivo, potentially mediated by BMAL1. These interventions to enhance therapy efficacy show preliminary feasibility, as pediatric leukemia patients responded favorable to preparation of macronutrient matched meals.

The Potential Transcriptomic and Metabolomic Mechanisms of ATO and ATRA in Treatment of FLT3-ITD Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) with Fms-like tyrosine kinase 3 gene internal tandem duplication (FLT3-ITD) mutations has a poor prognosis. The combination of arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) has a synergistic killing effect on leukemia cells with FLT3-ITD mutation. However, the mechanism, especially the changes of gene expression and metabolic activity remain unclear. Here we explore the transcriptome and metabolomics changes of FLT3-ITD AML cells treated with ATO/ATRA. RNA-seq was used to identify differential expressed genes (DEGs), and ultra-high performance liquid chromatography-quadrupole electrostatic field orbital trap mass spectrometry (UHPLC-QE-MS) nontargeted metabolomics method was used to screen out the differential metabolites in FLT3-ITD mutant cell lines treated with ATRA and ATO. KEGG pathway database was utilized for pathway exploration and Seahorse XF24 was used to detect extracellular acidification rate (ECAR). Metabolic polymerase chain reaction (PCR) array and real-time quantitative PCR (RT-qPCR) were used to detect mRNA levels of key metabolic genes of glycolysis and fatty acid after drug treatment. A total of 3873 DEGs were identified and enriched in 281 Gene Ontology (GO) terms, among which 210 were related to biological processes, 43 were related to cellular components, and 28 were related to molecular functions. Besides, 1794 and 927 differential metabolites were screened in positive and negative ion mode separately, and 59 different metabolic pathways were involved, including alanine-aspartate-glutamate metabolic pathway, arginine, and proline metabolic pathway, glycerophospholipid metabolic pathways, etc. According to KEGG Pathway analysis of transcriptome combined with metabolome, glycolysis/gluconeogenesis pathway and fatty acid metabolism pathway were significantly founded enriched. ATRA + ATO may inhibit the glycolysis of FLT3-ITD AML cells by inhibiting FLT3 and its downstream AKT/HK2-VDAC1 signaling pathway. The gene transcription profile and metabolites of FLT3-ITD mutant cells changes significantly after treatment, which might be related to the anti-FLT3-ITD AML effect. The screened DEGs, differential metabolites pathway are helpful in studying the mechanism of anti-leukemia effects and drug targets.

CALCRL knockdown suppresses cancer stemness and chemoresistance in acute myeloid leukemia with FLT3-ITD and DNM3TA-R882 double mutations.

Acute myeloid leukemia (AML)patients with FLT3 internal tandem duplication (FLT3-ITD)and DNA methyltransferase 3A (DNMT3A)R882 double mutations had a worse prognosis compared withAML with FLT3-ITDor DNMT3A R882 single mutation. This study was designed to explore the specific role of Calcitonin Receptor Like (CALCRL)in AML with FLT3-ITDand DNMT3A R882 double mutations. MOLM13 cells were transduced with CRISPR knockout sgRNA constructs to establish the FTL3-ITDand DNMT3A-R882double-mutated AML cell model. Quantitative real-time PCR and Western blot assay were carried out to examine corresponding gene and protein expression. Methylation of CALCRL promoter was measured by methylation-specific PCR (MSP).Cell viability, colony formation, flow cytometry, and sphere formation assays were conducted to determine cell proliferation, apoptosis, and stemness. MOLM13 cells were exposed to stepwise increasing concentrations of cytarabine (Ara-C) to generate MOLM13/Ara-Ccells. An in vivo AML animal model was established, and the tumor volume and weight were recorded. TUNEL assay was adopted to examine cell apoptosis in tumor tissues. DNMT3A-R882 mutation upregulated the expression of CALCRL while downregulated the DNA methylation level of CALCRL in MOLM13 cells. CALCRL knockdown greatly inhibited cell proliferation, promoted apoptosisand repressed cell stemness, accompanied with the downregulated Oct4, SOX2, and Nanog in DNMT3A-R882-mutated MOLM13 cells and MOLM13/Ara-C cells. Furthermore, CALCRL knockdown restricted tumor growth and the chemoresistance of AML in vivo, as well as inducing cell apoptosis in tumor tissues. Together, these data reveal that CALCRL is a vital regulator of leukemia cell survival and resistance to chemotherapy, suggesting CALCRL as a promising therapeutic target for the treatment of FTL3-ITDand DNMT3A-R882 double-mutated AML.

Prognostic risk signature in patients with acute myeloid leukemia treated with hypomethylating agents and venetoclax

AbstractHypomethylating agents (HMAs) and venetoclax (Ven) represent the standard of care for patients with acute myeloid leukemia (AML) who are ineligible for intensive chemotherapy. However, the European LeukemiaNet (ELN) risk classifications have been validated for patients treated with intensive therapy. In this study, we validate a recently proposed new molecular prognostic risk signature (mPRS) for patients with AML treated with HMAs and Ven. This classification allocated patients to favorable, intermediate (N/KRAS or FLT3–internal tandem duplication mutations), and lower (TP53 mutations) benefit groups. We retrospectively analyzed 159 patients treated with HMA and Ven. The mPRS classification allocated 74 (47%), 31 (19%), and 54 (34%) patients to the higher, intermediate, and lower-benefit groups, respectively. The overall response rate was 71% (86%, 54%, and 59% in the higher, intermediate, and lower-benefit groups, respectively). The median overall survival (OS) and event-free survival (EFS) times were 30 and 19 months, respectively, in the higher-benefit group; 12 and 8 months in the intermediate-benefit group; and 5 and 4 months in the lower-benefit group (P < .001). The C-index for OS and EFS was higher when stratifying patients according to mPRS classification than with the ELN 2022 classification. The 2-year cumulative incidence of relapse was 35%, 70%, and 60% in the higher, intermediate, and lower-benefit groups, respectively (P = .005). The mPRS classification accurately segregated groups of patients with AML treated with HMA plus Ven. In these patients, N/KRAS and TP53 mutations appear to negatively affect outcomes; therefore, new treatment approaches are warranted.

Clinical and morphological features of central nervous system tumor with &lt;I&gt;BCOR&lt;/I&gt; internal tandem duplication

In 2022, central nervous system (CNS) tumor with BCOR internal tandem duplication (BCOR ITD) was included in the fifth edition of the World Health Organization Classification of Tumors of the CNS as part of the embryonal tumor group. The identification a distinct DNA methylation profile and the presence of a recurrent genetic aberration – BCOR ITD – made it possible to recognize these tumors as a separate entity. In most cases, these tumors occur in children under 5 years of age and are located in the hemispheres of the cerebellum or brain. Since CNS tumor with BCOR ITD is a rare and relatively new tumor entity, it is not yet widely known and can be misdiagnosed. The most common initial diagnosis in patients referred for a second opinion to our reference center is anaplastic/classical medulloblastoma. In this article, we aimed to summarize the characteristic clinical, morphological and immunophenotypic features of CNS tumors with BCOR ITD based on 8 clinical cases confirmed by molecular genetic testing. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology.

Identification Of Tet2 Gene Level Of Expression In Acute Leukemias

Objective: TET2 gene (4q24) is one of the most important regulatory factors of the methylation process in human genome. The homologous protein is directly involved in convertion of 5-methylcytosine into 5-hydroximethylcytosine (1). Loss of function mutations in TET2 gene are considered to be driver mutations (initiating mutations) for leukemogenesis. In some cases they can be found in early clonal cells of Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL) or myelodisplastic syndromes (MDS) (2). The aim of this study is to determinate the frequency of TET2 mutations in Acute Leukemia (AL) patients. Materials and methods: This retrospective study enrolled 24 patients newly diagnosed with AML (19 patients) and ALL (5 pacients). By using Real-Time PCR we obtained a relative cuantification of TET2 gene level of expression taking ABL gene as reference. Outcomes: Low levels of TET2 gene expression were found in 6 patients (25%), 3 of them being diagnosed with AML and 3 with ALL. No cytogenetic anomalies were noticed. However, all these patients had blastocytosis and higher white blood cells count than pacients with normal levels of expression. There was one case with mutations in both TET2 gene and NPM1 gene and one case that associated a defective TET2 gene and an internal tandem duplication of FLT3-ITD. Conclusions: In our study, TET2 gene loss of function mutations occured on a specific background which involved some characteristic mutations for AL and some particular features of cell morphology. We aim to continue this study by investigating the long term effects of this epigenetic mutation in AL patients.