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.
Venetoclax plus azacitidine as genetic-driven bridge-to-transplant therapy for IDH2-mutated acute myeloid leukaemia (AML) refractory to intensive chemotherapy: proof-of-concept case reports Gaetano Cimino, Matteo Caridi, Valeria Cardinali, Sofia Sciabolacci, Sara De Santis, Camilla Rellini, Caterina Matteucci, Cristina Mecucci, Paolo Sportoletti, Francesco Zorutti, Alessandra Carotti, Roberta La Starza, Antonio Pierini, Maria Paola Martelli Annals of Hematology, 2025 Despite the greater biological understanding and the new drugs available, acute myeloid leukaemia (AML) patients who are refractory to intensive induction chemotherapy represents an unmet clinical need, especially in young/fit adults who are eligible for bone marrow transplantation. Since venetoclax/azacitidine (ven/aza) was introduced in AML management in 2020, survival of elderly/unfit patients has dramatically improved, especially in those carrying NPM1 or IDH2 mutations. However, the use of ven/aza in young and fit adults remains limited, raising ongoing debate about its potential role beyond patients ineligible to intensive chemotherapy. Here, we discuss three under 60 years chemorefractory AML patients, who, given the concomitant IDH2 mutations, were started to ven/aza as bridge-to-transplant and successfully treated. These cases confirm the extraordinary sensitivity of IDH2-mutated AML to aza/ven even in the refractoriness setting and show that such less-intensive regimen can be driven by genetics offering a promising alternative to intensive salvage chemotherapy, while preserving patient fitness for allo-transplant.
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, Dorian Forte, Jacopo Nanni, Martina Barone, Alessandra Grassi, Gianluca Cristiano, Claudia Venturi, Valentina Robustelli, Giulia Atzeni, Cristina Mosca, Sara De Santis, Cecilia Monaldi, Andrea Poletti, Carolina Terragna, Antonio Curti, Michele Cavo, Simona Soverini, Emanuela Ottaviani 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.
Unstable major molecular response as a trigger for next generation sequencing-based BCR::ABL1 mutation testing in chronic myeloid leukemia Adela Benesova, Sara De Santis, Vaclava Polivkova, Pavla Pecherkova, Jitka Krizkova, Pavla Suchankova, Cecilia Monaldi, Hana Klamova, Dana Srbova, Hana Zizkova, Andreas Hochhaus, Simona Soverini, Katerina Machova Polakova American Journal of Hematology, 2024 In chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKIs), molecular response (MR) milestones have been identified that harbor prognostic significance. Major molecular response (MMR)—defined as 3-log reduction in BCR::ABL1 transcript levels from the standardized baseline on the International Scale (IS)—was the very first of such MR milestones to be recognized: It emerged at the time of the IRIS trial as a “safe haven” protecting from loss of response and progression.1 According to the ELN recommendations, achievement of MMR after 12 months of TKI treatment defines an optimal response whereas failure to achieve MMR or loss of MMR represents a warning, requiring the therapeutic strategy to be carefully evaluated for continuation or change.2 Emerging TKI-resistant point mutations in the kinase domain (KD) of BCR::ABL1 may underlie warning responses in a proportion of patients, tilting the balance toward treatment change. Previous studies have interestingly shown that a peculiar kinetics of BCR::ABL1 transcripts fluctuating around 0.1% IS (“unstable MMR”) may be observed during TKI therapy in some patients and have suggested it may be an early indicator for BCR::ABL1 KD mutation testing.3 However, further data are needed to support inclusion of unstable MMR among the indications for mutation testing in clinical recommendations. The aim of this collaborative EUTOS (European Treatment and Outcome Study for CML) study was to take advantage of next generation sequencing (NGS) to investigate the role of BCR::ABL1 KD mutations in CML patients with unstable MMR on TKI therapy. In order to assess the clinical relevance of low frequency variants and the value of NGS-based analysis in this setting, we established a dedicated bioinformatic pipeline including an in-house developed software using a statistically derived dynamic threshold for mutation calling, which enabled us to maximize discrimination between true variants and sequencing errors. Importantly, we showed that therapy changes in patients with confirmed BCR::ABL1 mutations during unstable MMR resulted in higher probability of deep molecular response (DMR) achievement. A total of 91 CML patients with unstable MMR on TKI therapy were analyzed by NGS, with unstable MMR defined as BCR::ABL1 transcript levels in the range of 0.1% IS ±0.16 (the uncertainty of the measurement) for a minimum of 6 months. Two groups were identified according to molecular response trends (Table S1). Group A (n = 15/91) included patients on TKI therapy (1st line, n = 13; 2nd line, n = 1; 5th line, n = 1) displaying unstable MMR after an initial DMR. In regard to ELN recommendation, all patients were classified as warning.2 Group B (n = 76/91) consisted of patients (1st line, n = 59 [8/59 patients maintained an unstable MMR also during subsequent lines of therapy]; 2nd and/or subsequent line, n = 17) who had achieved no better response than unstable MMR. According to ELN recommendations, these patients have optimal response even when BCR::ABL1 levels fluctuate around the level of 0.1% IS. Detailed characteristics of the patients are summarized in Table S2. Follow-up samples were also analyzed. Altogether, 159 unstable MMR samples were analyzed by NGS (median, two samples per patient; range, 1–11) during the course of unstable MMR period. Sample preparation, BCR::ABL1 transcript quantification, NGS analysis, Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantification (LoQ) estimation and statistical analysis are described in detail in Supplementary data including Tables S3–S5, Figures S1–S3. Having optimized a bioinformatic pipeline minimizing NGS errors at low mutation frequencies (Supplementary data; Figure S4), we investigated the rate and kinetics of BCR::ABL1 KD variants in patients with unstable MMR. We chose to perform the analysis in duplicate (starting from reverse transcription of RNA to cDNA) to assess reproducibility of mutation results at lower transcript levels, and we checked for mutation kinetics in follow-up samples. Variant frequencies (VAFs) of variants detected in both duplicates were similar and we provided their mean. We found that underlying BCR::ABL1 KD mutations can be detected both in patients who display unstable MMR after an initial DMR (group A) and in patients with persistent unstable MMR as their best response (group B)—although mutations were more frequently observed in the former than in the latter group. The median VAF in both duplicates was 80% (range 4.6%–100%) in group A. In group B the median of VAF in both duplicates was 9.1% (range 4.4%–30%). Mutations were indeed found in 9/15 (60%) as against 12/76 (16%) patients, respectively (Figure 1); in 7/15 (47%) and 6/76 (8%) patients, the mutations identified could be recognized as poorly sensitive to the administered TKI based on IC50 data or literature reports. However, 3/9 mutations in group A and 7/12 mutations (one at high level) in group B were detected in only one of the two replicates. Except for two mutations (F311L and M244V), all low-level mutations (below 20% VAF) and also one high-level mutation detected in only one replicate were not confirmed in follow-up samples, despite no change in therapy. One possible explanation is that these mutations might have been artifacts. Even when using an optimized pipeline of NGS data analysis, in case of a single assessment some low-level variants may happen to pass the LoQ reaching the statistically significant VAF for calling. In contrast, variants passing this level in both duplicates are highly likely to be true mutations. However, it cannot be excluded that at low BCR::ABL1 transcripts and low mutation frequency, true mutations may happen to be picked in one replicate but not in another. Nevertheless, the fact that they disappeared in subsequent samples indicates that such few mutant molecules would have no clinical relevance anyway.4 To assess the frequency of low-level variants in relation to BCR::ABL1 transcript levels, 499 samples from 151 patients with transcript levels in the range of 0.1%–1% IS and 287 samples from 143 patients with transcript levels >1% IS routinely analyzed by NGS between 2017 and 2021 were compared. In addition, samples from 30 healthy donors were used for estimation of NGS error rates at each nucleotide position of the KD. Frequency of detected clinically relevant mutations did not differ between the samples with BCR::ABL1 transcript level >1% IS and with 0.1%–1% IS. However, low-level variants were significantly more frequent in samples with BCR::ABL1 transcript levels 0.1%–1% IS. The results are described in detail in Supplementary data including Tables S6 and S7. To evaluate the clinical benefit of mutational analysis during unstable MMR, the probability of DMR achievement was compared between patients with unchanged therapy and with therapy change (TKI switch or TKI dose increase) (Figure S5). In group A, the therapy change increased the probability of DMR re-achievement regardless of whether the patients had no BCR::ABL1 mutations (n = 3) or had confirmed mutations (n = 5). In contrast, DMR was not re-achieved in two patients with mutations and no therapy change. A decreased probability of DMR re-achievement was found also for patients without mutations and no therapy change (n = 5). The differences were not significant due to low number of patients. Similar observations were found for group B: a higher probability of DMR achievement was observed in patients who changed the therapy, both in case of confirmed mutations (n = 5) and in case of no mutations (n = 31). The probability of DMR was slightly lower for patient without mutations and without therapy change (n = 38), but was not significantly different compared to patients with therapy change (Figure S5). In conclusion, TKI switch or dose increase in patients with detected mutations during unstable MMR led to higher probability of DMR achievement compared to no therapy change. Based on these results, we propose unstable MMR as a novel trigger for BCR::ABL1 KD mutation analysis. Loss of DMR and BCR::ABL1 increase to the levels of unstable MMR is most probability associated with the development of TKI-resistant mutations; thus, once 0.1% IS BCR::ABL1 transcript level is reached, samples should undergo NGS analysis. For patients whose response to TKI therapy fluctuates around MMR with no further improvement, the likelihood of an underlying mutation is much lower, and NGS analysis might be performed 1–2 times per year during unstable MMR, and promptly in case of BCR::ABL1 transcript increase above the uncertainty of the measurement. The reasons of achievement of unstable MMR as the best response may be more often related to patient compliance, lower doses of TKIs, or clonal hematopoiesis. The loss of deep MR represents a risk factor of BCR::ABL1 resistant mutation development. Patients, whose measurable residual disease fluctuates at levels of MMR, but not deeper, are in lower risk of TKI-resistant mutation acquisition. We suggest the use of our novel bioinformatic tool (freely available at nextdom.uhkt.cz) specifically optimized for BCR::ABL1 KD mutation calling and calibrated on the calculation of error rates all over the KD of the BCR::ABL1 transcript. Based on our observations, mutation testing in unstable MMR samples should be done in duplicate. Moreover, whenever total BCR::ABL1 levels are in the range 0.1%–1% IS, dynamics of low-level mutations (below 20% VAF) should be checked in at least one subsequent sample before treatment change. AB performed mutational analysis, established the bioinformatical pipeline, evaluated data, drafted methodological part of the manuscript, and created Tables and Figures of the paper; SdS and CM performed mutational analysis; VP participated on mutational analysis; PP developed NextDOM; JK participated on mutational analysis; PS converted NextDOM to open access tool and performed statistical analysis; HK evaluated and provided clinical data; DS evaluated and provided clinical data; HZ was responsible for BCR::ABL1 transcript level monitoring; AH supervised the work and the project; SS coordinated the study, interpreted results, and revised the paper; KMP designed the study, interpreted results, and wrote the paper. This work was funded by Project Grant NU21-07-00225 from the Czech Health Research Council, by the European Treatment and Outcome study (EUTOS) for CML, by MH CZ – DRO (IHBT – 00023736), and by Ministerstvo Školství, Mládež a Tělovýchovy (MEYS CZ) (BBMRI.cz no. LM2023033). Computational resources were supplied by the project “e-Infrastruktura CZ” (e-INFRA CZ ID:90140) supported by MEYS CZ. The authors are grateful to the patients for providing their samples for this study. AH, KMP, and SS received support by Novartis through the European Treatment and Outcome Study (EUTOS) for CML. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1. Supporting Information FIGURE S1. Scheme of LoQ calculation based on sequenced ABL1 kinase domain of 30 healthy donors. (A) After data normalization the data were divided into individual nucleotide changes and using the Poisson distribution the limits “of specific nucleotide change” were counted; (B) After data normalization the number of reads of each position were processed with Poisson distribution to obtain the limits “of each nucleotide position.” FIGURE S2. The threshold data set cover the range of amino acids A28-D504. The amplicons prepared for sequencing are delineated with full arrow: for the patient sample (PT—upper part) it is 1493 bp, for the healthy donor (HD) it is 1429 bp. The threshold data set was generated for the region 272–1704 (1433 bp), that is, A28-D504 (ref. seq. NM_005157) and is depicted with dashed arrow. FIGURE S3. The limits of quantification of the most frequently detected TKI-resistant mutations (n = 29). Red bars show the most frequently used uniform thresholds levels in published NGS studies—1% and 3%. The T315I mutation is highlighted with red color. FIGURE S4. The NextDOM pipeline. (A) Scheme depicts how the software NextDOM utilizes the data generated in software NextGENe, furthermore the data are normalized and evaluated using either LoD or LoQ data set. As a final outcome, the statistically significant variants are provided (p-value ≤.05). (B) The interphase of NextDOM comprises of windows for paths to files and of instructions for using either LoD or LoQ data set. FIGURE S5. Cumulative achievement of DMR of patients with therapy change and with unchanged therapy in relation of mutation detection during unstable MMR. (A) Analysis of patients from Group A. (B) Analysis of patients from Group B. Blue curve—patients with therapy change during unstable MMR with no mutation detected; dashed blue curve—patients with therapy change during unstable MMR with mutation detected; red curve—patients no therapy changes during unstable MMR with no mutation detected; dashed red—patients no therapy changes during unstable MMR with mutation detected. TABLE S1. Characteristics of CML patients with unMMR during TKI therapy. Group A includes patients who displayed unMMR after an initial DMR. Group B includes patients who never achieved any better response than unMMR. TABLE S2. Patient characteristics. Group A includes patients (n = 15) on TKI therapy who had initially achieved deep molecular response, but BCR::ABL1 transcripts had subsequently increased to the levels of unMMR. Group B consists of patients (n = 76) who never achieved better response to TKIs than unMMR TABLE S3. The LoQ and thresholds of significant mutation detection. TABLE S4. The values of LoD and LoQ for each specific nucleotide change. (A) Thirty samples of healthy donors were sequenced and for each type of nucleotide change the LoDs were calculated. (B) The values of LoQs were set to fivefold the LoDs. TABLE S5. Limits of quantification of each nucleotide position. LoQs were calculated for 1248 positions of the KD, and for each position they were compared to the value of LoQ of the specific nucleotide changes. Then the higher value was chosen for the final threshold data set. The second column shows the total number of each individual nucleotide in the 1248 bp region. The third and the fifth columns show the number of nucleotides with higher LoQ for each nucleotide positions for transitions and for transversion, respectively. TABLE S6. Frequency of low- and high-level variants detected by NGS in samples with BCR::ABL1 below or above 1% IS. TABLE S7. ddASO PCR analysis of low-level variants. The table presents the results of ddASO PCR analysis of five low-level variants, which were detected by NGS only in one duplicate, in five samples. Only in one case the variant was confirmed by ddASO PCR. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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, Chiara Sartor, Samantha Bruno, Livio Pagano, Marianna Criscuolo, Roberta Zanotti, Massimiliano Bonifacio, Patrizia Tosi, Michel Arock, Peter Valent, Michele Cavo, Simona Soverini 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, Cecilia Monaldi, Sara De Santis, Nicoletta Testoni, Simona Soverini, Gianantonio Rosti, Michele Cavo, Fausto Castagnetti 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.
Decreased bone mineral density in Costello syndrome Chiara Leoni, David A. Stevenson, Lucilla Martini, Roberto De Sanctis, Giovanna Mascolo, Francesca Pantaleoni, Sara De Santis, Ilaria La Torraca, Silvia Persichilli, Paolo Caradonna, Marco Tartaglia, Giuseppe Zampino Molecular Genetics and Metabolism, 2014
Publications
FOXM1 TRANSCRIPTION FACTOR OVER-EXPRESSION IN CHRONIC MYELOID LEUKEMIA: A NEW COMPONENT OF BCR-ABL1+ LEUKEMIC STEM CELL PERSISTENCE UNDER TYROSINE KINASE INHIBITOR THERAPY. 10.1002/
HYPER-ACTIVATION OF AURORA KINASE A-POLO-LIKE KINASE 1-FOXM1 AXIS PROMOTES CHRONIC MYELOID LEUKEMIA RESISTANCE TO TYROSINE KINASE INHIBITORS. 10.1186/s13046-019-1197-9.
