AUTOPHAGY INHIBITION POTENTIATES MIDOSTAURIN EFFICACY IN FLT3-MUTATED ACUTE MYELOID LEUKEMIA UNDER HYPOXIC CONDITIONS Cristina Mosca Haematologica, 2026 Mutations in FMS-like tyrosine kinase 3 (FLT3) are found in about 30% of newly diagnosed acute myeloid leukemia (AML) patients and confer poor prognosis. Despite the multikinase inhibitor midostaurin (mido) has improved outcomes for FLT3-mutated (FLT3m) AML, relapses occur in over 40% of cases. The hypoxic bone marrow niche provides a protective environment for leukemic stem cells (LSCs), playing a crucial role in the development of resistance mechanisms. We investigated in vitro how hypoxia impacts on the efficacy of mido on FLT3m AML cells and how therapeutic efficacy of mido can be enhanced.Mido and/or chloroquine (Cq) were administered as single agents and in combination to FLT3-ITD AML cell lines MV-4-11 and MOLM-13 under normoxic (20% O2) and hypoxic (1% O2) conditions. Apoptosis induction, cell proliferation, metabolic activity, gene expression changes and protein expression were evaluated after 24 and 48 hours of exposure to the drugs.In FLT3-mutated AML cell lines, the main metabolic alteration due to mido treatment was a reduction in glutamate levels. Gene expression analysis revealed lower transcriptional levels of genes encoding enzymes involved in non-essential amino acid synthesis and m-TORC1 signalling suppression. These results suggests that autophagy was promoted as a potential survival mechanism to evade the antileukemic effects of mido. Co-treatment with the autophagy inhibitor Cq enhanced apoptosis and reduced expression of phosphorylated FLT3 and autophagy-related proteins (BECLIN, ATG3, LC3I-II) under both normoxic and hypoxic conditions. According to autophagy inhibition, the combination reversed Mido-induced mTORC1 suppression and ultimately activated DNA repair pathways, including ATM and p53 signalling.Our data suggest that Cq might contribute to eradicate FLT3m cells in hypoxia. The association of mido with autophagy inhibitors could be evaluated as a therapeutic strategy to overcome LSC resistance in the hypoxic niche.Supported by the AIRC-Fellowship for Italy, Project Code: 26986.
INTEGRATION OF MULTIPLE RNA-SEQ DATASETS ENHANCES DEEP PROFILING OF NPM1-MUTATED FLT3-ITD NEGATIVE AML Matteo Caridi Haematologica, 2026 Introduction. Acute Myeloid Leukemia (AML) is an aggressive, molecularly heterogeneous malignancy with limited survival despite advances in its characterization and treatment. Nucleophosmin (NPM1) mutations without FLT3-ITD (NPM1mut, FLT3-ITDneg) define a subgroup with favorable outcomes using chemotherapy alone. However, up to 40% of these patients relapse. Attempts to refine prognosis through additional gene mutations have failed. We therefore deeply analyzed the biological landscape of this clinically relevant subgroup using an integrated RNA-seq dataset, aiming to identify novel prognostic and biological biomarkers, including through machine-learning (ML) approaches.Methods. Three public and one unpublished RNA-seq datasets were included in the study. We created the 'Gu' algorithm (EHA 2025 - PS1463) to generate an n = 894 unified RNA-seq dataset with strong biological coherence. Synthetic data for data augmentation purposes were generated via the "synthpop" R package, performing a 300× augmentation that yielded n = 2,682 synthetic samples. Several bioinformatic and ML analyses were performed to investigate the clinically relevant NPM1mut FLT3-ITDneg AML subgroup. Results: The unified dataset demonstrated correct data integration and biological coherence (Figure 1a), also reproducing the ELN 2022 risk stratification (Fig. 1b). Within NPM1-mutated AML (n = 308, 187 FLT3-wt), the transcriptomic profile was heterogeneous, contrasting with homogeneous entities such as APL or t(8;21) AML. The NPM1-mut signature was confirmed, with HOX genes upregulated and CD34/CD133 downregulated (Figure 1c); new genes related to NPM1mut were identified. Focusing on the NPM1mut FLT3-ITDneg AML, a distinct transcriptomic signature based on FLT3-ITD mutational status was identified (Figure 1d) beside a group of genes that was stably upregulated in NPM1mut independently of FLT3, allowing definition of an NPM1-mut “core” (mainly HOX genes). Transcriptomic dissection revealed two subgroups (Figure 1e): a “FLT3-like” cluster resembling FLT3-ITD AML, and an “NPM1-pure” cluster enriched in inflammatory/immune genes. RAS mutations prevailed in FLT3-like and IDH2 in NPM1-pure cases. No survival difference emerged under standard therapy. Finally, genes with prognostic value were identified, including genes related to immune regulation. A scoring system based on the expression of a 4-gene-panel could effectively stratify prognosis (Fig 1f). Validations are ongoing.Conclusions: Our work opened an innovative window into the biology of NPM1-mutated FLT3-wild-type AML, revealing a distinctive transcriptomic signature that had never emerged from previous analyses, as limited sample size had so far precluded deep investigations on this subgroup. Several potential biomarkers for further prognostic stratification and new targets for therapy have emerged. Biological validations are ongoing in our lab.
SETD2 LOSS IN CHRONIC MYELOID LEUKEMIA PROMOTES METABOLIC REPROGRAMMING AND GENOMIC INSTABILITY LEADING TO THERAPEUTIC RESISTANCE AND DISEASE PROGRESSION Manuela Mancini Haematologica, 2026 Chronic myeloid leukemia (CML) is driven by the Ph chromosome, but additional genetic abnormalities (AGAs) such as ASXL1 mutations and other cytogenetic alterations contribute to poor prognosis and reduced treatment response. SETD2 plays a critical role in DNA repair and chromatin integrity and its non genomic loss-of-function (LOF) has been linked to disease progression in CML. This study examines how SETD2 LOF impacts on CML pathogenesis and its potential as a biomarker for high-risk disease.To investigate the role of SETD2 LOF in CML, cellular models with SETD2 silencing or overexpression were compared using liquid chromatography-tandem mass spectrometry (LC-MS/MS), RNA sequencing (RNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq). Validation was performed by Western blotting (WB), immunofluorescence (IF), and co-immunoprecipitation (co-IP) in proper cell fractions. Additionally, SNP-array analysis provided genomic insights.Differential transcriptomic profiling revealed SETD2-dependent transcriptional regulation of genes involved in DNA repair (MSH2, MSH6) and metabolic homeostasis (PFKP, LDHA, PDK1) (FIGURE 1A). Differential interactome profiling by LC-MS/MS identified SETD2 interactions with proteins critically involved in mismatch repair (MSH2, MSH6), cell division (α-/β-tubulin), and glycolysis (PFKP, PFKFB3, PD, and LDHA). Notably, SETD2 was also found to interact with key kinases regulating proliferation and stress response, including ERK1/2 and p38 MAPK. Integration of SNP-array analysis after chronic cell exposure to DNA damaging agents with ChIP-seq of SETD2-dependent H3K36me3 deposition sites confirmed that SETD2 plays a direct role in promoting faithful DNA damage response, since breakpoints were enriched at sites where H3K36me3 was disrupted by SETD2 LOF.Notably, we uncovered a novel role for SETD2 LOF in rewiring cellular metabolism: SETD2 re-expression attenuated the glycolytic shift observed in SETD2-deficient cells, as evidenced by downregulation of glycolytic enzymes (FIGURE 1 B, C, D), mitochondrial oxidative phosphorylation and overexpression of IDH1 enzyme which regulates a key step in the TCA, produces NADPH and protects cells from reactive oxygen species, acting as an antioxidant. Finally, SETD2/H3K36me3 deficiency as assessed by WB in total leukocytes could be detected in pts with AGAs at diagnosis and could discriminate pts who subsequently achieved non-optimal vs optimal responses to IM.Our findings point to SETD2 LOF as a key cooperating event in CML, that may act since diagnosis to set the stage for TKI resistance and disease acceleration by:• sustaining BCR::ABL1-independent genomic instability that fuels the acquisition of AGAs• metabolic reprogramming towards glycolysisWhether SETD2 LOF may serve as a biomarker of high-risk disease at diagnosis is an intriguing hypothesis that we are currently exploring in a larger cohort of uniformly treated pts.
Tracking Response and Resistance in Acute Myeloid Leukemia through Single-Cell DNA Sequencing Helps Uncover New Therapeutic Targets Samantha Bruno, Enrica Borsi, Agnese Patuelli, Lorenza Bandini, Manuela Mancini, et al. International Journal of Molecular Sciences, 2024 Acute myeloid leukemia (AML) is an aggressive hematologic neoplasia with a complex polyclonal architecture. Among driver lesions, those involving the FLT3 gene represent the most frequent mutations identified at diagnosis. The development of tyrosine kinase inhibitors (TKIs) has improved the clinical outcomes of FLT3-mutated patients (Pt). However, overcoming resistance to these drugs remains a challenge. To unravel the molecular mechanisms underlying therapy resistance and clonal selection, we conducted a longitudinal analysis using a single-cell DNA sequencing approach (MissionBioTapestri® platform, San Francisco, CA, USA) in two patients with FLT3-mutated AML. To this end, samples were collected at the time of diagnosis, during TKI therapy, and at relapse or complete remission. For Pt #1, disease resistance was associated with clonal expansion of minor clones, and 2nd line TKI therapy with gilteritinib provided a proliferative advantage to the clones carrying NRAS and KIT mutations, thereby responsible for relapse. In Pt #2, clonal architecture was less complex, and 1st line TKI therapy with midostaurin was able to eradicate the leukemic clones. Our results corroborate previous findings about clonal selection driven by TKIs, highlighting the importance of a deeper characterization of individual clonal architectures for choosing the best treatment plan for personalized approaches aimed at optimizing outcomes.
SETD2 non genomic loss of function in advanced systemic mastocytosis is mediated by an Aurora kinase A/MDM2 axis and can be therapeutically targeted Manuela Mancini, Cecilia Monaldi, Sara De Santis, Cristina Papayannidis, Michela Rondoni, et al. Biomarker Research, 2023 Background The SETD2 tumor suppressor gene encodes a histone methyltransferase that safeguards transcription fidelity and genomic integrity via trimethylation of histone H3 lysine 36 (H3K36Me3). SETD2 loss of function has been observed in solid and hematologic malignancies. We have recently reported that most patients with advanced systemic mastocytosis (AdvSM) and some with indolent or smoldering SM display H3K36Me3 deficiency as a result of a reversible loss of SETD2 due to reduced protein stability. Methods Experiments were conducted in SETD2-proficient (ROSAKIT D816V) and -deficient (HMC-1.2) cell lines and in primary cells from patients with various SM subtypes. A short interfering RNA approach was used to silence SETD2 (in ROSAKIT D816V cells), MDM2 and AURKA (in HMC-1.2 cells). Protein expression and post-translational modifications were assessed by WB and immunoblotting. Protein interactions were tested by using co-immunoprecipitation. Apoptotic cell death was evaluated by flow cytometry after annexin V and propidium iodide staining, respectively. Drug cytotoxicity in in vitro experiments was evaluated by clonogenic assays. Results Here, we show that the proteasome inhibitors suppress cell growth and induce apoptosis in neoplastic mast cells by promoting SETD2/H3K36Me3 re-expression. Moreover, we found that Aurora kinase A and MDM2 are implicated in SETD2 loss of function in AdvSM. In line with this observation, direct or indirect targeting of Aurora kinase A with alisertib or volasertib induced reduction of clonogenic potential and apoptosis in human mast cell lines and primary neoplastic cells from patients with AdvSM. Efficacy of Aurora A or proteasome inhibitors was comparable to that of the KIT inhibitor avapritinib. Moreover, combination of alisertib (Aurora A inhibitor) or bortezomib (proteasome inhibitor) with avapritinib allowed to use lower doses of each drug to achieve comparable cytotoxic effects. Conclusions Our mechanistic insights into SETD2 non-genomic loss of function in AdvSM highlight the potential value of novel therapeutic targets and agents for the treatment of patients who fail or do not tolerate midostaurin or avapritinib.
Prognosis in Chronic Myeloid Leukemia: Baseline Factors, Dynamic Risk Assessment and Novel Insights Miriam Iezza, Sofia Cortesi, Emanuela Ottaviani, Manuela Mancini, Claudia Venturi, et al. Cells, 2023 The introduction of tyrosine kinase inhibitors (TKIs) has changed the treatment paradigm of chronic myeloid leukemia (CML), leading to a dramatic improvement of the outcome of CML patients, who now have a nearly normal life expectancy and, in some selected cases, the possibility of aiming for the more ambitious goal of treatment-free remission (TFR). However, the minority of patients who fail treatment and progress from chronic phase (CP) to accelerated phase (AP) and blast phase (BP) still have a relatively poor prognosis. The identification of predictive elements enabling a prompt recognition of patients at higher risk of progression still remains among the priorities in the field of CML management. Currently, the baseline risk is assessed using simple clinical and hematologic parameters, other than evaluating the presence of additional chromosomal abnormalities (ACAs), especially those at “high-risk”. Beyond the onset, a re-evaluation of the risk status is mandatory, monitoring the response to TKI treatment. Moreover, novel critical insights are emerging into the role of genomic factors, present at diagnosis or evolving on therapy. This review presents the current knowledge regarding prognostic factors in CML and their potential role for an improved risk classification and a subsequent enhancement of therapeutic decisions and disease management.
