Ex Vivo Anti-Leukemic Effect of Exosome-like Grapefruit-Derived Nanovesicles from Organic Farming-The Potential Role of Ascorbic Acid.

Citrus fruits are a natural source of ascorbic acid, and exosome-like nanovesicles obtained from these fruits contain measurable levels of ascorbic acid. We tested the ability of grapefruit-derived extracellular vesicles (EVs) to inhibit the growth of human leukemic cells and leukemic patient-derived bone marrow blasts. Transmission electron microscopy and nanoparticle tracking analysis (NTA) showed that the obtained EVs were homogeneous exosomes, defined as exosome-like plant-derived nanovesicles (ELPDNVs). The analysis of their content has shown measurable amounts of several molecules with potent antioxidant activity. ELPDNVs showed a time-dependent antiproliferative effect in both U937 and K562 leukemic cell lines, comparable with the effect of high-dosage ascorbic acid (2 mM). This result was confirmed by a clear decrease in the number of AML blasts induced by ELPDNVs, which did not affect the number of normal cells. ELPDNVs increased the ROS levels in both AML blast cells and U937 without affecting ROS storage in normal cells, and this effect was comparable to ascorbic acid (2 mM). With our study, we propose ELPDNVs from grapefruits as a combination/supporting therapy for human leukemias with the aim to improve the effectiveness of the current therapies.

Retinoic acid and proteotoxic stress induce AML cell death overcoming stromal cell protection.

BACKGROUND: Acute myeloid leukemia (AML) patients bearing the ITD mutation in the tyrosine kinase receptor FLT3 (FLT3-ITD) present a poor prognosis and a high risk of relapse. FLT3-ITD is retained in the endoplasmic reticulum (ER) and generates intrinsic proteotoxic stress. We devised a strategy based on proteotoxic stress, generated by the combination of low doses of the differentiating agent retinoic acid (R), the proteasome inhibitor bortezomib (B), and the oxidative stress inducer arsenic trioxide (A). METHODS: We treated FLT3-ITD+ AML cells with low doses of the aforementioned drugs, used alone or in combinations and we investigated the induction of ER and oxidative stress. We then performed the same experiments in an in vitro co-culture system of FLT3-ITD+ AML cells and bone marrow stromal cells (BMSCs) to assess the protective role of the niche on AML blasts. Eventually, we tested the combination of drugs in an orthotopic murine model of human AML. RESULTS: The combination RBA exerts strong cytotoxic activity on FLT3-ITD+ AML cell lines and primary blasts isolated from patients, due to ER homeostasis imbalance and generation of oxidative stress. AML cells become completely resistant to the combination RBA when treated in co-culture with BMSCs. Nonetheless, we could overcome such protective effects by using high doses of ascorbic acid (Vitamin C) as an adjuvant. Importantly, the combination RBA plus ascorbic acid significantly prolongs the life span of a murine model of human FLT3-ITD+ AML without toxic effects. Furthermore, we show for the first time that the cross-talk between AML and BMSCs upon treatment involves disruption of the actin cytoskeleton and the actin cap, increased thickness of the nuclei, and relocalization of the transcriptional co-regulator YAP in the cytosol of the BMSCs. CONCLUSIONS: Our findings strengthen our previous work indicating induction of proteotoxic stress as a possible strategy in FLT3-ITD+ AML therapy and open to the possibility of identifying new therapeutic targets in the crosstalk between AML and BMSCs, involving mechanotransduction and YAP signaling.

YY1 Knockdown Relieves the Differentiation Block and Restores Apoptosis in AML Cells.

In this study we analyzed the expression of Yin and Yang 1 protein (YY1), a member of the noncanonical PcG complexes, in AML patient samples and AML cell lines and the effect of YY1 downregulation on the AML differentiation block. Our results show that YY1 is significantly overexpressed in AML patient samples and AML cell lines and that YY1 knockdown relieves the differentiation block. YY1 downregulation in two AML cell lines (HL-60 and OCI-AML3) and one AML patient sample restored the expression of members of the CEBP protein family, increased the expression of extrinsic growth factors/receptors and surface antigenic markers, induced morphological cell characteristics typical of myeloid differentiation, and sensitized cells to retinoic acid treatment and to apoptosis. Overall, our data show that YY1 is not a secondary regulator of myeloid differentiation but that, if overexpressed, it can play a predominant role in myeloid differentiation block.

How acute myeloid leukemia (AML) escapes from FMS-related tyrosine kinase 3 (FLT3) inhibitors? Still an overrated complication?

FMS-related tyrosine kinase 3 (FLT3) mutations, present in about 25%-30% of acute myeloid leukemia (AML) patients, constitute one of the most frequently detected mutations in these patients. The binding of FLT3L to FLT3 activates the phosphatidylinositol 3-kinase (PI3K) and RAS pathways, producing increased cell proliferation and the inhibition of apoptosis. Two types of FLT3 mutations exist: FLT3-ITD and FLT3-TKD (point mutations in D835 and I836 or deletion of codon I836). A class of drugs, tyrosine-kinase inhibitors (TKI), targeting mutated FLT3, is already available with 1st and 2nd generation molecules, but only midostaurin and gilteritinib are currently approved. However, the emergence of resistance or the selection of clones not responding to FLT3 inhibitors has become an important clinical dilemma, as the duration of clinical responses is generally limited to a few months. This review analyzes the insights into mechanisms of resistance to TKI and poses a particular view on the clinical relevance of this phenomenon. Has resistance been overlooked? Indeed, FLT3 inhibitors have significantly contributed to reducing the negative impact of FLT3 mutations on the prognosis of AML patients who are no longer considered at high risk by the European LeukemiaNet (ELN) 2022. Finally, several ongoing efforts to overcome resistance to FLT3-inhibitors will be presented: new generation FLT3 inhibitors in monotherapy or combined with standard chemotherapy, hypomethylating drugs, or IDH1/2 inhibitors, Bcl2 inhibitors; novel anti-human FLT3 monoclonal antibodies (e.g., FLT3/CD3 bispecific antibodies); FLT3-CAR T-cells; CDK4/6 kinase inhibitor (e.g., palbociclib).

Applicability of 2022 classifications of acute myeloid leukemia in the real-world setting.

The increasing knowledge of molecular genetics of acute myeloid leukemia (AML) necessitated the update of previous diagnostic and prognostic schemes, which resulted in the development of the World Health Organization (WHO), the International Consensus Classification (ICC), and the new European LeukemiaNet (ELN) recommendations in 2022. We aimed to provide a real-world application of the new models, unravel differences and similarities, and test their implementation in clinical AML diagnosis. A total of 1001 patients diagnosed with AML were reclassified based on the new schemes. The overall diagnostic changes between the WHO 2016 and the WHO 2022 and ICC classifications were 22.8% and 23.7%, respectively, with a 13.1% difference in patients' distribution between ICC and WHO 2022. The 2022 ICC "not otherwise specified" and WHO "defined by differentiation" AML category sizes shrank when compared with that in WHO 2016 (24.1% and 26.8% respectively, vs 38.7%), particularly because of an expansion of the myelodysplasia (MDS)-related group. Of 397 patients with a MDS-related AML according to the ICC, 55.9% were defined by the presence of a MDS-related karyotype. The overall restratification between ELN 2017 and ELN 2022 was 12.9%. The 2022 AML classifications led to a significant improvement of diagnostic schemes. In the real-world setting, conventional cytogenetics, usually rapidly available and less expensive than molecular characterization, stratified 56% of secondary AML, still maintaining a powerful diagnostic role. Considering the similarities between WHO and ICC diagnostic schemes, a tentative scheme to generate a unified model is desirable.

MCL1 regulates AML cells metabolism via direct interaction with HK2. Metabolic signature at onset predicts overall survival in AMLs' patients.

We characterize the metabolic background in distinct Acute Myeloid Leukemias (AMLs), by comparing the metabolism of primary AML blasts isolated at diagnosis with that of normal hematopoietic maturing progenitors, using the Seahorse XF Agilent. Leukemic cells feature lower spare respiratory (SRC) and glycolytic capacities as compared to hematopoietic precursors (i.e. day 7, promyelocytes). According with Proton Leak (PL) values, AML blasts can be grouped in two well defined populations. The AML group with blasts presenting high PL or high basal OXPHOS plus high SRC levels had shorter overall survival time and significantly overexpressed myeloid cell leukemia 1 (MCL1) protein. We demonstrate that MCL1 directly binds to Hexokinase 2 (HK2) on the outer mitochondrial membrane (OMM). Overall, these results suggest that high PL and high SRC plus high basal OXPHOS levels at disease onset, arguably with the concourse of MCL1/HK2 action, are significantly linked with shorter overall survival time in AML. Our data describe a new function for MCL1 protein in AMLs' cells: by forming a complex with HK2, MCL1 co-localizes to VDAC on the OMM, thus inducing glycolysis and OXPHOS, ultimately conferring metabolic plasticity and promoting resistance to therapy.

Acute Myeloid Leukemia with NPM1 Mutation and Disseminated Leukemia Cutis: Achievement of Molecular Complete Remission by Venetoclax/Azacitidine Combination in a Very Old Patient.

We describe a case of acute myeloid leukemia with NPM1 mutation and disseminated leukemia cutis in a very old patient, who achieved a long-lasting response to the azacitidine/venetoclax combination with molecular complete remission, given the potential value of this rarely observed clinical outcome.

Clinical significance of mitochondrial DNA content in acute promyelocytic leukaemia.

Although a growing body of evidence demonstrates that altered mtDNA content (mtDNAc) has clinical implications in several types of solid tumours, its prognostic relevance in acute promyelocytic leukaemia (APL) patients remains largely unknown. Here, we show that patients with higher-than-normal mtDNAc had better outcomes regardless of tumour burden. These results were more evident in patients with low-risk of relapse. The multivariate Cox proportional hazard model demonstrated that high mtDNAc was independently associated with a decreased cumulative incidence of relapse. Altogether, our data highlights the possible role of mitochondrial metabolism in APL patients treated with ATRA.

Genomic landscape of patients with FLT3-mutated acute myeloid leukemia (AML) treated within the CALGB 10603/RATIFY trial.

The aim of this study was to characterize the mutational landscape of patients with FLT3-mutated acute myeloid leukemia (AML) treated within the randomized CALGB 10603/RATIFY trial evaluating intensive chemotherapy plus the multi-kinase inhibitor midostaurin versus placebo. We performed sequencing of 262 genes in 475 patients: mutations occurring concurrently with the FLT3-mutation were most frequent in NPM1 (61%), DNMT3A (39%), WT1 (21%), TET2 (12%), NRAS (11%), RUNX1 (11%), PTPN11 (10%), and ASXL1 (8%) genes. To assess effects of clinical and genetic features and their possible interactions, we fitted random survival forests and interpreted the resulting variable importance. Highest prognostic impact was found for WT1 and NPM1 mutations, followed by white blood cell count, FLT3 mutation type (internal tandem duplications vs. tyrosine kinase domain mutations), treatment (midostaurin vs. placebo), ASXL1 mutation, and ECOG performance status. When evaluating two-fold variable combinations the most striking effects were found for WT1:NPM1 (with NPM1 mutation abrogating the negative effect of WT1 mutation), and for WT1:treatment (with midostaurin exerting a beneficial effect in WT1-mutated AML). This targeted gene sequencing study provides important, novel insights into the genomic background of FLT3-mutated AML including the prognostic impact of co-mutations, specific gene-gene interactions, and possible treatment effects of midostaurin.

Molecular dissection of a hyper-aggressive CBFB-MYH11/FLT3-ITD-positive acute myeloid leukemia.

Acute Myeloid Leukaemia (AML) is a haematological malignancy showing a hypervariable landscape of clinical outcomes and phenotypic differences, explainable by heterogeneity at the cellular and molecular level. Among the most common genomic alterations, CBFB-MYH11 rearrangement and FLT3-ITD gene mutations, have opposite clinical significance and are unfrequently associated. We present here a Molecular Case Report in which these two events co-exist an ultra-aggressive phenotype resulting in death in 4 days from hospital admittance. Somatic and germline Whole Exome Sequencing analysis was performed to uncover other putative driver mutations, de-novo genomic structural events or germline clusters increasing cancer insurgence. Only three mutations in LTK, BCAS2 and LGAS9 were found, unlikely causative of the exhibited phenotype, prompting to additional investigation of the rare CBFB-MYH11/ FLT3-ITD scenario.

An Polycythemia Vera Evolve from Acute Myeloid Leukemia? Report of a Case Showing a Simultaneous Minor JAK2 V617F Mutated Clone.

The evolution of myeloproliferative neoplasms (MPN) to acute myeloid leukemia (AML) occurs in 2-10% of patients, depending on the MPN subtype, treatment, and follow-up length. The reverse-path from AML to MPN has been rarely reported. We herein present a 75 years old woman with AML, in whom a JAK2-V617F positive polycythemia vera (PV) emerged during follow-up, 19 months from the end of consolidation treatment. JAK2-V617F mutation screening retrospectively performed by Next Generation Sequencing (NGS) and JAK2 MutaScreen was negative on the bone marrow sample collected at AML diagnosis. However, using digital droplet PCR (ddPCR), we detected a minor JAK2 V617F mutated clone at AML onset. In addition, a TET2 R550 mutated clone persisted at stable levels throughout the disease course. This case shows that a very small MPN clone masked at AML diagnosis may expand after treatment end and be erroneously interpreted as MPN evolving from AML. Very sensitive techniques such as ddPCR may help to unravel the true disease history in these cases.

Vitamin C Deficiency in Patients With Acute Myeloid Leukemia.

Vitamin C has been shown to play a significant role in suppressing progression of leukemia through epigenetic mechanisms. We aimed to study the role of vitamin C in acute myeloid leukemia (AML) biology and clinical course. To this purpose, the plasma levels of vitamin C at diagnosis in 62 patients with AML (including 5 cases with acute promyelocytic leukemia, APL),7 with myelodysplastic syndrome (MDS), and in 15 healthy donors (HDs) were studied. As controls, vitamins A and E levels were analysed. Expression of the main vitamin C transporters and of the TET2 enzyme were investigated by a specific RQ-PCR while cytoplasmic vitamin C concentration and its uptake were studied in mononuclear cells (MNCs), lymphocytes and blast cells purified from AML samples, and MNCs isolated from HDs. There were no significant differences in vitamin A and E serum levels between patients and HDs. Conversely, vitamin C concentration was significantly lower in AML as compared to HDs (p<0.0001), inversely correlated with peripheral blast-counts (p=0.029), significantly increased at the time of complete remission (CR) (p=0.04) and further decreased in resistant disease (p=0.002). Expression of the main vitamin C transporters SLC23A2, SLC2A1 and SLC2A3 was also significantly reduced in AML compared to HDs. In this line, cytoplasmic vitamin C levels were also significantly lower in AML-MNCs versus HDs, and in sorted blasts compared to normal lymphocytes in individual patients. No association was found between vitamin C plasma levels and the mutation profile of AML patients, as well as when considering cytogenetics or 2017 ELN risk stratification groups. Finally, vitamin C levels did not play a predictive role for overall or relapse-free survival. In conclusion, our study shows that vitamin C levels are significantly decreased in patients with AML at the time of initial diagnosis, further decrease during disease progression and return to normal upon achievement of CR. Correspondingly, low intracellular levels may mirror increased vitamin C metabolic consumption in proliferating AML cells.

Prevalence and Prognostic Role of IDH Mutations in Acute Myeloid Leukemia: Results of the GIMEMA AML1516 Protocol.

IDH1/2 mutations are common in acute myeloid leukemia (AML) and represent a therapeutic target. The GIMEMA AML1516 observational protocol was designed to study the prevalence of IDH1/2 mutations and associations with clinico-biological parameters in a cohort of Italian AML patients. We analyzed a cohort of 284 AML consecutive patients at diagnosis, 139 females and 145 males, of a median age of 65 years (range: 19−86). Of these, 38 (14%) harbored IDH1 and 51 (18%) IDH2 mutations. IDH1/2 mutations were significantly associated with WHO PS >2 (p < 0.001) and non-complex karyotype (p = 0.021) when compared to IDH1/2-WT. Furthermore, patients with IDH1 mutations were more frequently NPM1-mutated (p = 0.007) and had a higher platelet count (p = 0.036). At relapse, IDH1/2 mutations were detected in 6 (25%) patients. As per the outcome, 60.5% of IDH1/2-mutated patients achieved complete remission; overall survival and event-free survival at 2 years were 44.5% and 36.1%, respectively: these rates were similar to IDH1/2-WT. In IDH1/2-mutated patients, high WBC proved to be an independent prognostic factor for survival. In conclusion, the GIMEMA AML1516 confirms that IDH1/2 mutations are frequently detected at diagnosis and underlines the importance of recognizing IDH1/2-mutated cases up-front to offer the most appropriate therapeutic strategy, given the availability of IDH1/2 inhibitors.

Ascorbate Plus Buformin in AML: A Metabolic Targeted Treatment.

In the present study, we characterized the metabolic background of different Acute Myeloid Leukemias' (AMLs) cells and described a heterogeneous and highly flexible energetic metabolism. Using the Seahorse XF Agilent, we compared the metabolism of normal hematopoietic progenitors with that of primary AML blasts and five different AML cell lines. We assessed the efficacy and mechanism of action of the association of high doses of ascorbate, a powerful oxidant, with the metabolic inhibitor buformin, which inhibits mitochondrial complex I and completely shuts down mitochondrial contributions in ATP production. Primary blasts from seventeen AML patients, assayed for annexin V and live/dead exclusion by flow cytometry, showed an increase in the apoptotic effect using the drug combination, as compared with ascorbate alone. We show that ascorbate inhibits glycolysis through interfering with HK1/2 and GLUT1 functions in hematopoietic cells. Ascorbate combined with buformin decreases mitochondrial respiration and ATP production and downregulates glycolysis, enhancing the apoptotic effect of ascorbate in primary blasts from AMLs and sparing normal CD34+ bone marrow progenitors. In conclusion, our data have therapeutic implications especially in fragile patients since both agents have an excellent safety profile, and the data also support the clinical evaluation of ascorbate-buformin in association with different mechanism drugs for the treatment of refractory/relapsing AML patients with no other therapeutic options.

The Venetoclax/Azacitidine Combination Targets the Disease Clone in Acute Myeloid Leukemia, Being Effective and Safe in a Patient with COVID-19.

The addition of Venetoclax (VEN) to Hypomethylating agents (HMAs) significantly improves the probability of complete remission and prolongs survival in patients with Acute Myeloid Leukemia (AML) when compared to HMA alone. However, the mutated clone composition may impact the probability of response and its duration. Here, we describe the molecular profile of a patient with AML rapidly evolved from a previous therapy-related-Chronic MyeloMonocytic Leukemia, who achieved safely complete remission after treatment with the VEN/Azacitidine combination, even in the presence of SARS-COVID-2 infection. The targeted NGS analysis showed that the VEN/AZA combination led to the eradication of the FLT3-ITD and RUNX1 mutated clone/s primarily associated with AML evolution, and subsequently, the SRSF2, NRAS, and ASXL1 mutated clone/s. This case also underlines the importance of the sequential use of targeted NGS for disease monitoring: the deep molecular remission achieved by this patient allowed to safely guide adjustments of drug dosage and treatment intervals in the presence of neutropenia, helping to rule out disease progression.

Atypical Rearrangements in APL-Like Acute Myeloid Leukemias: Molecular Characterization and Prognosis.

Acute promyelocytic leukemia (APL) accounts for 10-15% of newly diagnosed acute myeloid leukemias (AML) and is typically caused by the fusion of promyelocytic leukemia with retinoic acid receptor α (RARA) gene. The prognosis is excellent, thanks to the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) combination therapy. A small percentage of APLs (around 2%) is caused by atypical transcripts, most of which involve RARA or other members of retinoic acid receptors (RARB or RARG). The diagnosis of these forms is difficult, and clinical management is still a challenge for the physician due to variable response rates to ATRA and ATO. Herein we review variant APL cases reported in literature, including genetic landscape, incidence of coagulopathy and differentiation syndrome, frequent causes of morbidity and mortality in these patients, sensitivity to ATRA, ATO, and chemotherapy, and outcome. We also focus on non-RAR rearrangements, complex rearrangements (involving more than two chromosomes), and NPM1-mutated AML, an entity that can, in some cases, morphologically mimic APL.

Clinical relevance of an objective flow cytometry approach based on limit of detection and limit of quantification for measurable residual disease assessment in acute myeloid leukemia. A post-hoc analysis of the GIMEMA AML1310 trial.

Using a multiparametric flow cytometry assay, we assessed the predictive power of a threshold calculated applying the criteria of limit of detection (LOD) and limit of quantitation (LOQ) in adult patients with acute myeloid leukemia. This was a post-hoc analysis of 261 patients enrolled in the GIMEMA AML1310 prospective trial. According to the protocol design, using the predefined measurable residual disease (MRD) threshold of 0.035% bone marrow residual leukemic cells (RLC) calculated on mononuclear cells, 154 (59%) of the 261 patients were negative (MRD <0.035%) and 107 (41%) were positive (MRD ≥0.035%). Using LOD and LOQ, we selected the following categories of patients: (i) LODneg if RLC were below the LOD (74; 28.4%); (ii) LODpos-LOQneg if RLC were between the LOD and LOQ (43; 16.5%); and (iii) LOQpos if RLC were above the LOQ (144; 54.4%). Two-year overall survival of these three categories of patients was 75.4%, 79.8% and 66.4%, respectively (P=0.1197). Given their superimposable outcomes, the LODneg and LODpos-LOQneg categories were combined. Two-year overall survival of LODneg/LODpos-LOQneg patients was 77.0% versus 66.4% of LOQpos individuals (P=0.043). This figure was challenged in univariate analysis (P=0.046, hazard ratio=1.6, 95% confidence interval: 1.01-2.54) which confirmed the independent role of the LOD-LOQ approach in determining overall survival. In the AML1310 protocol, using the threshold of 0.035%, 2-year overall survival of patients with MRD <0.035% and MRD ≥0.035% was 74.5% versus 66.4%, respectively (P=0.3521). In conclusion, the use of the LOD-LOQ method results in more sensitive detection of MRD that, in turn, translates into a more accurate recognition of patients with different outcomes.