Extramedullary (EM) colonization is a rare complication of acute myeloid leukemia (AML), occurring in about 10% of patients, but the processes underlying tissue invasion are not entirely characterized. Through the application of RNAseq technology, we examined the transcriptome profile of 13 AMLs, 9 of whom presented an EM localization. Our analysis revealed significant deregulation within the extracellular matrix (ECM)-receptor interaction and focal-adhesion pathways, specifically in the EM sites. The transcription factor TWIST1, which is known to impact on cancer invasion by dysregulating epithelial–mesenchymal-transition (EMT) processes, was significantly upregulated in EM-AML. To test the functional impact of TWIST1 overexpression, we treated OCI-AML3s with TWIST1 -siRNA or metformin, a drug known to inhibit tumor progression in cancer models. After 48 h, we showed downregulation of TWIST1 , and of the EMT-related genes FN1 and SNAI2 . This was associated with significant impairment of migration and invasion processes by Boyden chamber assays. Our study shed light on the molecular mechanisms associated with EM tissue invasion in AML, and on the ability of metformin to interfere with key players of this process. TWIST1 may configure as candidate marker of EM-AML progression, and inhibition of EMT-pathways may represent an innovative therapeutic intervention to prevent or treat this complication.

Abstract Purpose: We evaluated leukemia-associated immunophenotypes (LAIP) and their correlation with fms-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) gene mutational status in order to contribute a better identification of patients at highest risk of relapse in acute myeloid leukemia (AML). Experimental Design: Bone marrow samples from 132 patients with AML were analyzed by nine-color multiparametric flow cytometry. We confirmed the presence of the mutation in diagnostic samples and in sorted cells by conventional RT-PCR and by patient-specific RQ-PCR. Results: Within the CD34+ cell fraction, we identified a discrete population expressing high levels of the IL3 receptor α-chain (CD123) and MIC-2 (CD99) in combination with the IL2 receptor α-chain (CD25). The presence of this population positively correlated with the internal tandem duplications (ITD) mutation in the FLT3 gene (r = 0.71). Receiver operating characteristics showed that, within the CD34+ cell fraction a percentage of CD123/CD99/CD25+ cells ≥11.7% predicted FLT3–ITD mutations with a specificity and sensitivity of >90%. CD34/CD123/CD99/CD25+ clones were also detectable at presentation in 3 patients with FLT3 wild-type/NPM1+ AML who relapsed with FLT3-ITD/NPM1+ AML. Quantitative real-time PCR designed at relapse for each FLT3-ITD in these three cases confirmed the presence of low copy numbers of the mutation in diagnostic samples. Conclusions: Our results suggest that the CD34/CD25/CD123/CD99+ LAIP is strictly associated with FLT3-ITD–positive cells. Clin Cancer Res; 21(17); 3977–85. ©2015 AACR.

Background: Although acute promyelocytic leukemia (APL) is the most favorable subtype of acute myeloid leukemia, it remains a life-threating disease for high-risk patients. Despite progress in APL with arsenic (ATO) plus retinoic acid (RA) as front-line treatment, approximately 10-15% of patients will acquire resistance to ATO and/or RA and relapse. Relapse after ATO/RA based therapy remains very challenging, and novel drug schemes to overcome resistance are urgently needed. Analysis of bone marrow (BM) biopsies of 15 consecutive APL patients at diagnosis, revealed a homogeneous staining for BCL2 in all cases. These results suggest BCL2 inhibition could be a therapeutic option for resistant APL. In vitro treatment with the BCL2 inhibitor Venetoclax (VEN), resulted in increased MCL1 expression, suggesting a resistance mechanism to this therapy. Aims: Here, we investigated the effects of VEN+MCL1 inhibitor S64315 in APL cells resistant to ATO/RA, and evaluated the effect of the combination therapy in vivo, using a cell line derived xenograft (CLDX) model for APL with the ATO-resistant associated mutation PML A216V. Methods: Primary leukemic blasts from APL mice (n=4) and patients (age, 25-52y; n=10) were treated with VEN (250-500nM), ATO, ATRA (1µM), and S64315 (10-50nM) monotherapies and combinations and evaluated for cell survival. In addition, we evaluated the transcriptome of 16 APL patients (age, 25-74y; 7 males) enrolled in the TCGA study regarding the BCL2 expression. NB4/NB4R2 (RA-resistant) cell lines were transduced with PML A216V. Additionally, U937 cells were transduced with the inducible PML-RARA system. After induction of PML-RARA expression with Zinc for 0-24h, BCL2 protein levels were evaluated by western blot. Additionally, RA/ATO treated (1µM) NB4 cells for 6-72h were evaluated for BCL2/MCL1 levels. For the CLDX model, NB4 PML A216V cells were transplanted into NSG mice, and after chimerism detection (hCD45+) in the peripheral blood, mice were equally distributed into 4 groups of treatment: vehicle, VEN (25mg/kg/day), S64315 (20mg/Kg/day) and combination (n=5/group) for 21 consecutive days. Results: Gene set enrichment analysis using the transcriptome of APL patients with BCL2high associated these patients with the terms “EPPERT_HSC_LSC” and “17_LSC”. These results suggest that APL patients with high BCL2 expression differ in their metabolic and proliferative state compared to patients with BCL2low. Functionally, the induction of PML-RARA expression in U937 cells resulted in increased BCL2 expression in a time-dependent manner (peak at 6h). Contrarily, treatment with ATO/RA resulted in decreased BCL2 levels with upregulation of the MCL1 protein. BH3 profile revealed that ATO/RA treated NB4 cells displayed an increase in the MCL1 anti-apoptotic dependency. Additionally, in vitro and ex vivo treatment of APL cells (cell lines and primary blasts) revealed synergic activity of S64315, but not the BCL2 inhibitor (VEN), in combination with ATO. Furthermore, treatment of ATO resistant NB4 cells (by gradual exposure to ATO or by expression of the PML A216V) revealed sensitivity of those cells to the combination VEN+S64315. Finally, in vivo CLDX model revealed limited effect of VEN and S64315 monotherapy, while the combination was able to effectively reduce the leukemic burden, resulting in increased overall survival (vehicle:15 vs Combo:42 days). Summary/Conclusion: The combination of BCL2 and MCL1 inhibition is a potential effective treatment in ATO/RA resistant APL patients and could be clinically explored in this category of patients with very poor prognosis.

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.

Data derived from high-throughput sequencing technologies have allowed a deeper understanding of the molecular landscape of Acute Myeloid Leukemia (AML), paving the way for the development of novel therapeutic options, with a higher efficacy and a lower toxicity than conventional chemotherapy. In the antileukemia drug development scenario, ascorbic acid, a natural compound also known as Vitamin C, has emerged for its potential anti-proliferative and pro-apoptotic activities on leukemic cells. However, the role of ascorbic acid (vitamin C) in the treatment of AML has been debated for decades. Mechanistic insight into its role in many biological processes and, especially, in epigenetic regulation has provided the rationale for the use of this agent as a novel anti-leukemia therapy in AML. Acting as a co-factor for 2-oxoglutarate-dependent dioxygenases (2-OGDDs), ascorbic acid is involved in the epigenetic regulations through the control of TET (ten-eleven translocation) enzymes, epigenetic master regulators with a critical role in aberrant hematopoiesis and leukemogenesis. In line with this discovery, great interest has been emerging for the clinical testing of this drug targeting leukemia epigenome. Besides its role in epigenetics, ascorbic acid is also a pivotal regulator of many physiological processes in human, particularly in the antioxidant cellular response, being able to scavenge reactive oxygen species (ROS) to prevent DNA damage and other effects involved in cancer transformation. Thus, for this wide spectrum of biological activities, ascorbic acid possesses some pharmacologic properties attractive for anti-leukemia therapy. The present review outlines the evidence and mechanism of ascorbic acid in leukemogenesis and its therapeutic potential in AML. With the growing evidence derived from the literature on situations in which the use of ascorbate may be beneficial in vitro and in vivo, we will finally discuss how these insights could be included into the rational design of future clinical trials.

Background: Myelofibrosis (MF) displays the worst prognosis among Philadelphia-negative myeloproliferative neoplasms and is characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and frequent transformation to acute myeloid leukemia. Different therapeutic approaches are being used depending on the severity and the specific clinical manifestations of the disease in each patient. Unfortunately, no curative therapy is currently available for MF, except for bone marrow transplantation, which however has a consistent percentage of failure. There is therefore a great urgency to identify biomarkers correlated with the different stages of the disease and to treat patients with a more tailored treatment. Long non-coding RNAs (lncRNAs) have been recently described as key mediators in the development of hematological malignancies. Moreover, circulating lncRNAs have already been proposed as a new class of non-invasive biomarkers for cancer diagnosis and prognosis. Aims: The aim of this study was to identify circulating lncRNAs whose plasmatic concentration differs between MF patients and healthy donors (HDs). Subsequently, we evaluated their potential role as non-invasive disease biomarkers in MF. Methods: As a preliminary result we analyzed the expression of 38 lncRNAs in CD34+ cells collected from 83 MF patients and 26 HDs leading to the identification of 26 differentially expressed lncRNAs. To confirm these results and to move to a more accessible sample type we collected plasma from 143 MF patients and 65 HDs. RNA was extracted from these samples and the relative abundance of lncRNAs, including some of those deregulated in CD34+ cells or already described as involved in hematological malignancies and myeloid differentiation, was assessed using qRT-PCR. According to the plasmatic levels of each lncRNA patients were split into two groups (low- or high-) and this subdivision was used to unveil the potential correlation between the various lncRNAs and the clinical features of MF patients. Results: Our analysis identified 7 lncRNAs significantly upregulated in MF patients’ plasma compared to HDs. Among these, high levels of LINC01268, MALAT1 or GAS5 correlated with several detrimental clinical features of MF, such as high counts of leukocytes and CD34+ cells, a severe grade of bone marrow fibrosis and the presence of splenomegaly. Strikingly, high plasma levels of LINC01268 (Log-rank p-value = 0.0018), GAS5 (Log-rank p-value = 0.0008) or MALAT1 (Log-rank p-value = 0.0348) were associated with a poor overall-survival (OS) while high levels of LINC01268 correlated also with a shorter leukemia-free-survival (LFS). Finally, multivariate analysis demonstrated that a high plasma concentration of LINC01268 was an independent prognostic variable for both OS (HR = 2.104; confidence interval (CI) = 1.08–4.12; p = 0.0297) and LFS (HR = 8.190; CI = 1.02–65.78; p = 0.0479). Summary/Conclusion: To our knowledge, this is the first study describing the expression profile of circulating lncRNAs in MF patients’ plasma and focusing on their putative role as biomarkers in clinical practice. In particular, our results demonstrated that increased levels of circulating LINC01268, GAS5 or MALAT1 are associated with disease detrimental features and correlate with an inferior OS in MF patients. Notably, multivariate analysis confirmed that LINC01268 plasma levels might improve the identification of patients with a poor prognosis. If the prognostic value of this lncRNA will be confirmed in independent patients’ cohorts it might be used to integrate contemporary prognostic models.

(MF) is the Philadelphia-negative myeloproliferative neoplasm characterized by the worst prognosis and poor response to conventional therapy. Driver mutations in JAK2 and CALR impact on JAK-STAT pathway activation but also on the production of reactive oxygen species (ROS). ROS play a pivotal role in inflammation-induced oxidative damage to cellular components including DNA, that leads to greater genomic instability and promotes cell transformation. stress status in MF cells compared to JAK2 variant. Furthermore, plasma samples from CALR mutated patients have significantly lower TAC levels leading to a lower responsiveness to oxidative injury. On the other hand, increased TAC levels correlate with the presence of JAK2 mutation and several detrimental clinical features. MF patients with high plasmatic TAC display inferior survival and multivariate analysis demonstrated that increased TAC activity might represent a novel prognostic biomarker independent from DIPSS classification. We speculated that the high increase in oxidative stress in CALRmutated patients could be involved in the activation of protective mechanisms which ultimately promote cell death. On the contrary, in JAK2 mutated patients, the slight increase in oxidative stress can determine the persistence of cells with damaged DNA where the accumulation of mutations promotes the disease progression.

Background: Metabolic reprogramming is a basic feature in cancer. Leukemia associated cellular reprogramming, metabolic heterogeneity at onset and metabolic clonal evolution, driven by therapy, are essential insights that still remain unclear Aims: We compared Acute Myeloid Leukemias’ (AMLs) cells metabolism to hematopoietic progenitors’’ and normal maturing bone marrow cells’, to acquire useful prognostic information and to uncover actionable therapeutic targets. Methods: Methods We analyzed fresh primary blast from 19 AML patients, hematopoietic progenitors obtained from normal CD34+ cells differentiated to promyelocytes and granulocyte and the MV4-11, OCI-AML2 and OCI-AML3 cell lines, using a Seahorse Bioscience XFe96 analyzer. We silence MCL1 by siRNA and evaluated the interaction between HK2, MCL-1 and VDAC by co-immunoprecipitation and confocal microscopy. We assessed expression of MCL-1 and HK2 by western blot; HK2 by q-RTPCR. Results: Primary AML blast cells feature a lower spare respiratory capacity (SRC) (p=0.02) and lower glycolytic capacity (p=0.02) as compared to early progenitors/precursors (EP/P) from cultured CB CD34+ cells at day 7 of culture (N7, mostly promyelocytes) (Figure 1 a and b). Primary AML blast depend principally on fatty acids (p<0.05); they display a great flexibility, switching to glucose or glutamine to meet their energetic needs (Figure 1c). Consistent with the high adaptability of AML cells, and the emergence of resistance to therapy. We could define two populations (cut off value 10 pmol/min/x105 cells): one with higher (22±12 pmol/min/x105 cells) and on with lower (3±2 pmol/min/x105 cells) p<0.0001 levels of proton leak. The cases with higher proton leaks levels presented a reduced, extremely short, overall survival (p=0.048). We defined two SRC populations (cut off value 80 pmol/min/x105 cells): higher (124±47 pmol/min/x105 cells) and lower (52±25 pmol/min/x105cells) levels (p=0.0001). Interestingly the cases with higher SRC showed a trend of reduced overall survival. Associating high proton leak levels with high SRC the significance increases to p= 0.007. Considering the Basal OXPHOS of the AML patient’s cells, we observed two populations higher (54±12 pmol/min/x105 cells) and lower (16±6 pmol/min/x105cells) levels (p=0.0001). In patients with both high SRC plus high basal OXPHOS, the overall survival shortage is significant (p=0.002), indicating that an high SRC associated to higher basal respiration in AML blast cells confers greater aggressiveness and resistance to the therapy (Figure 1d). Patients with high mitochondrial respiration had a significantly higher myeloid cell leukemia 1 protein (MCL1) expression. We ascertained that MCL1 directly binds to Hexokinase II (HK2) on the outer mitochondrial membrane (OMM) affecting its stability. Image:Summary/Conclusion: We demonstrate that high proton leak and high mitochondrial respiration at onset, arguably with the concourse of MCL1/HK2 action, is significantly linked with a shorter overall survival in AMLs’ patients. Our data describe a new function of MCL1 protein in AMLs’ cells, forming a complex with HK2 co-localized to the voltage dependent anion channel (VDAC) on the OMM, thus promoting glycolysis and OXPHOS, ultimately conferring metabolic plasticity and promoting resistance to therapy. The lower spare respiratory capacity and lower glycolytic capacity of AML patients’ blast respect to normal early hematopoietic precursors suggest a therapeutic window to use glycolytic and mitochondrial inhibitors in resistant AML patients.