PML/RARA inhibits expression of HSP90 and its target AKT.

Essential for cell survival, the 90 kD Heat Shock Proteins (HSP90) are molecular chaperons required for conformational stabilization and trafficking of numerous client proteins. Functional HSP90 is required for the stability of AKT, a serine-threonine kinase phosphorylated in response to growth factor stimulation. AKT plays a crucial regulatory role in differentiation, cell cycle, transcription, translation, metabolism and apoptosis. Acute promyelocytic leukaemia (APL) is characterized by the presence of the promyelocytic leukaemia/retinoic acid receptor alpha (PML/RARA) fusion protein, which deregulates expression of several genes involved in differentiation and apoptosis. Here, we report inhibition of HSP90AA1 and HSP90AB1 isomer transcription in blasts isolated from patients with APL, associated with reduction of HSP90 protein expression and loss of control on AKT protein phosphorylation. We show that in vitro treatment of PML/RARA expressing cells with all-trans retinoic acid (ATRA) up-regulates HSP90 expression and stabilizes AKT. Addition of the HSP90-inhibitor 17-(allylamino)-17-demethoxygeldanamycin in combination with ATRA, blocks upregulation of AKT protein, indicating that HSP90 is necessary for ATRA action on AKT. This is the first report proving that expression of HSP90 isomers are directly and differentially repressed by PML/RARA, with critical results on cellular homeostasis of target proteins, such as AKT, in APL blasts.

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.

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.

NPM1 Mutated, BCR-ABL1 Positive Myeloid Neoplasms: Review of the Literature.

Breakpoint cluster region - Abelson (BCR-ABL1) chimeric protein and mutated Nucleophosmin (NPM1) are often present in hematological cancers, but they rarely coexist in the same disease. Both anomalies are considered founder mutations that inhibit differentiation and apoptosis, but BCR-ABL1 could act as a secondary mutation conferring a proliferative advantage to a pre-neoplastic clone. The 2016 World Health Organization (WHO) classification lists the provisional acute myeloid leukemia (AML) with BCR-ABL1, which must be diagnosed differentially from the rare blast phase (BP) onset of chronic myeloid leukemia (CML), mainly because of the different therapeutic approach in the use of tyrosine kinase inhibitors (TKI). Here we review the BCR/ABL1 plus NPMc+ published cases since 1975 and describe a case from our institution in order to discuss the clinical and molecular features of this rare combination, and report the latest acquisition about an occurrence that could pertain either to the rare AML BCR-ABL1 positive or the even rarer CML-BP with mutated NPM1 at the onset. Differential diagnosis is based on careful analysis of genotypic and phenotypic features and anamnestic, clinical evolution, and background data. Therapeutic decisions must consider the broader clinical aspects, the comparatively mild effects of TKI therapy versus the great benefit that might bring to most of the patients, as may be incidentally demonstrated by our case history.

PML/RARa Interferes with NRF2 Transcriptional Activity Increasing the Sensitivity to Ascorbate of Acute Promyelocytic Leukemia Cells.

: NRF2 (NF-E2 p45-related factor 2) orchestrates cellular adaptive responses to stress. Its quantity and subcellular location is controlled through a complex network and its activity increases during redox perturbation, inflammation, growth factor stimulation, and energy fluxes. Even before all-trans retinoic acid (ATRA) treatment era it was a common experience that acute promyelocytic leukemia (APL) cells are highly sensitive to first line chemotherapy. Since we demonstrated how high doses of ascorbate (ASC) preferentially kill leukemic blast cells from APL patients, we aimed to define the underlying mechanism and found that promyelocytic leukemia/retinoic acid receptor α (PML/RARa) inhibits NRF2 function, impedes its transfer to the nucleus and enhances its degradation in the cytoplasm. Such loss of NRF2 function alters cell metabolism, demarcating APL tissue from both normal promyelocytes and other acute myeloide leukemia (AML) blast cells. Resistance to ATRA/arsenic trioxide (ATO) treatment is rare but grave and the metabolically-oriented treatment with high doses of ASC, which is highly effective on APL cells and harmless on normal hematopoietic stem cells (HSCs), could be of use in preventing clonal evolution and in rescuing APL-resistant patients.

PYRROLO[1,2-b][1,2,5]BENZOTHIADIAZEPINES (PBTDs) induce apoptosis in K562 cells.

BACKGROUND: The objective of this study was to gain insight into the molecular mechanism of induced cell death (apoptosis) by PYRROLO [1,2-b][1,2,5]BENZOTHIADIAZEPINES (PBTDs) series compounds, using human (K562) cells as a model. METHODS: We focused our attention on some members of the PBTDs family to test their potential apoptotic activity in K562 cells. Important apoptotic activity was demonstrated, as evidenced by the concentration and percentage of cell death quantified by measuring PI-uptake by flow cytometry, and DNA fragmentation analyzed by agarose gel electrophoresis, generating a characteristic ladder pattern of discontinuous DNA fragments. The expression of Bcl-2 family was tested using western blotting and transfection method. RESULTS: PBTDs-mediated suppression of K562 cell proliferation was induced by apoptosis characterized by the appearance of DNA fragmentation and was associated with the poly(ADP-ribose)polymerase (PARP) cleavage. PBTD-1 and -3 treatment resulted in caspase-3 activation through down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we used K562/vector and K562/bcl-2 cells, which were generated by transfection of the cDNA of the Bcl-2 gene. As compared with K562/vector, K562/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment with 10 muM PBTD-1 and -3 for 24 h produced morphological features of apoptosis and DNA fragmentation in K562/vector cells, respectively. In contrast, PBTD-1 and -3-induced caspase-3 activation and apoptosis were inhibited in K562/Bcl-2. Furthermore, Bcl-2 overexpressing cells exhibited less cytocrome c release during PBTDs-induced apoptosis. CONCLUSION: These results indicate that PBTDs effectively induce apoptosis of K562 leukemia cells through the activation of caspase cascades. In addition, these findings indicate that Bcl-2 inhibits PBTD-1 and -3 induced-apoptosis via a mechanism that interferes with cytocrome c release, and the activity of caspase-3, which is involved in the execution of apoptosis.

High-dose ascorbate and arsenic trioxide selectively kill acute myeloid leukemia and acute promyelocytic leukemia blasts in vitro.

The use of high-dose ascorbate (ASC) for the treatment of human cancer has been attempted several decades ago and has been recently revived by several in vitro and in vivo studies in solid tumors. We tested the cytotoxic effects of ASC, alone or in combination with arsenic trioxide (ATO) in acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL). Leukemic cell lines and primary blasts from AML and APL patients were treated with graded concentrations of ASC, alone or in association with standard concentration (1 µM) of ATO. The ASC/ATO combination killed myeloid blasts, including leukemic CD34+ cells, while sparing CD34+ progenitors obtained from normal cord blood and bone marrow. Actually, approximately one-third (11/36) of primary AML cases were highly sensitive to the ASC/ATO combination. The mechanism of cell killing appeared to be related to increased oxidative stress and overproduction of ROS in a non-quantitative fashion, which resulted in induction of apoptosis. These effects were reverted by the addition of the antioxidant N-Acetyl-Cysteine (NAC). In the APL NB4 model, ASC induced direct degradation of the PML and PML/RARA proteins via caspase activation, while the transcriptional repressor DAXX was recruited in re-constituted PML nuclear bodies. Our findings encourage the design of pilot studies to explore the potential clinical benefit of ASC alone or in combination with ATO in advanced AML and APL.

Pyrrolo[1,2-b][1,2,5]benzothiadiazepines (PBTDs): A new class of agents with high apoptotic activity in chronic myelogenous leukemia K562 cells and in cells from patients at onset and who were imatinib-resistant.

Pyrrolo[1,2-b][1,2,5]benzothiadiazepine 5,5-dioxides (PBTDs) induced apoptosis in human BCR-ABL-expressing leukemia cells. The apoptotic activity was also observed in primary leukemic blasts, obtained from chronic myelogenous leukemia (CML) patients at onset or from patients in blast crisis and who were imatinib-resistant. Compounds 5 and 14 induced apoptosis before BCR-ABL protein expression and tyrosin phosphorylation were affected and activated different caspases in the apoptotic pathway. PBTDs are a new class of valid candidates for the treatment of CML.

A case of SRSF2 mutation in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by extremely variable clinical course indicating substantial differences in the biology of the disease. Molecular characterization provides new insights useful for treatment decision making. We report on a patient diagnosed with CLL, whose disease was characterized by episodes of rapid progression and disease stabilization, and in which a SRSF2 gene mutation was identified in the absence of other commonly known mutations of CLL. To the best of our knowledge this is the first case of SRSF2 gene mutation ever reported in CLL.

PML/RARa inhibits PTEN expression in hematopoietic cells by competing with PU.1 transcriptional activity.

Acute promyelocitic leukemia (APL) is characterized by the pathognomonic presence in leukemic blasts of the hybrid protein PML/RARA, that acts as a transcriptional repressor impairing the expression of genes that are critical to myeloid differentiation. Here, we show that primary blasts from APL patients express lower levels of the oncosuppressor protein PTEN, as compared to blast cells from other AML subtypes or normal bone marrow, and demonstrate that PML-RARA directly inhibits PTEN expression. We show that All-Trans Retinoic Acid (ATRA) triggers in APL cells an active chromatin status at the core regulatory region of the PTEN promoter, that allows the binding of the myeloid-regulating transcription factor PU.1, and, in turn, the transcriptional induction of PTEN. ATRA, via PML/RARA degradation, also promotes PTEN nuclear re-localization and decreases expression of the PTEN target Aurora A kinase. In conclusion, our findings support the notion that PTEN is one of the primary targets of PML/RARA in APL.

Development of a high-resolution melting curve analysis screening test for SRSF2 splicing factor gene mutations in myelodysplastic syndromes.

Somatic mutations of the spliceosome machinery have been recently identified by whole genome analysis in hematologic diseases, such as myelodysplastic syndrome, chronic lymphocytic leukemia, myeloproliferative neoplasms, acute myeloid leukemia, and advanced forms of mastocytosis, and also in nonhematologic conditions. SRSF2 is a member of the serine/arginine-rich family pre-mRNA splicing factors that plays a role in mRNA export from the nucleus and translation. We describe a high-resolution melting (HRM) curve analysis to screen for SRSF2 hotspot mutations in a fast, sensitive, and reliable way. Fifty bone marrow samples from patients with myelodysplastic syndrome were analyzed by the HRM assay and by direct sequencing. HRM screening identified four melting patterns corresponding to a negative (wild-type) group and three different mutated groups. Each mutated group was identified according to the positive control used: P95H, P95L, and P95R, respectively. An HRM mutated pattern was identified in seven patients. Positive and negative results from HRM were compared with direct sequencing results with a sensitivity and specificity of 100% (95% CI, 0.56-1, and 95% CI, 0.89-1, respectively). Analytical sensitivity analysis revealed a detection threshold of up to 1:9 (mutated/wild type) dilution. This rapid screening method may provide useful information for clinical decision making and be helpful to optimize laboratory resources and reduce turnaround time.

Possibility of long-term remission in patients with advanced hematologic malignancies after reduced intensity conditioning regimen (RIC) and allogeneic stem cell transplantation.

High-dose chemotherapy and radiation used as a preparative regimen for allogeneic stem cell transplantation (SCT) produces a considerable morbidity and mortality. An alternative strategy, developed to reduce transplant-related toxicity and to induce graft versus malignancy effect in the presence of full hematopoietic engraftment, includes the application of RIC that provide sufficient immunosuppression. We studied the efficacy and toxicity of RIC followed by allogeneic SCT in elderly patients or with relative contraindications to conventional SCT. In all, 22 patients with hematologic malignancies and HLA-identical sibling donors were included in this study. All patients were either refractory to therapy or beyond first complete remission (CR). The majority of patients received fludarabine 120 mg/m(2)+thiotepa 10 mg/kg as conditioning regimen and cyclosporine as graft versus host disease (GVHD) prophylaxis. Organ toxicity was acceptable and all evaluable patients achieved engraftment. Three patients (15%) showed grade >II aGVHD; extensive chronic GVHD occurred in three out of 15 evaluable patients (20%). With median follow-up of 63.5 months, survival was 31%, disease-free survival (DFS) was 23%. All the durable responses occurred in patients who developed GVHD. Transplant related mortality (TRM) at 100 days was 27.3%, 67% of that caused by infections, while 6-year cumulative incidence of TRM was 38%. Our data show that RIC: (1). allows the engraftment of HLA-matched hematopoietic stem cells (2). provide durable responses in patients not eligible for conventional SCT exploiting the graft-versus-malignancy effect and (3). is loaded by an high rate of fatal infections.

Positive selection of CD34+ cells by immunoadsorption: factors affecting the final yield and hematopoietic recovery in patients with hematological malignancies and solid tumors.

There is a progressive increase in the use of selected hematopoietic progenitor cells after myeloablative therapy in patients affected by malignancies. Our goal was to determine which blood parameters, in the starting cell population, influence the concentration of CD34+ progenitors and the removal of unwanted cells in the final product. Also, we evaluated the hematopoietic recovery and toxicity associated with peripheral blood stem cell infusion. We retrospectively reviewed 53 procedures of positive selection of CD34+ cells, performed with the Ceprate SC immunoadsorption system, in 47 paticnts affected by various hematologic malignancies and solid tumors. An increased percentage of CD34+ cells in the starting fraction was associated both with the final purity and enrichment of CD34+ cells and with a decreased percentage of CD3+ and CD19+ cells in the final product. A low platelet count before selection had a borderlinc influence on the recovery of CD34+ cells. Forty patients received a median of 5 x 10(6) CD34+ cells per kg; the absolute neutrophil count (ANC) reached 0.5 x 10(9)/l in a median of 10 days whereas a PLT count above 20 x 10(9)/l was observed in 14 days. The reinfusion of selected CD34+ cells, containing a very low amount of dymethylsulfoxide. was well tolerated and no adverse reactions were observed. Autologous transplantation with selected CD34+ cells is a safe and well-tolerated procedure in patients affected by hematologic malignancies and solid tumors. Positive selection of CD34+ cells seems to be related to the quality of the apheresis products, particularly to the initial CD34+ cell and PLT content.

The Management of Membranous Glomerulopathy in Allogeneic Stem Cells Transplantation: Updated Literature

BACKGROUND: membranous glomerulopathy (MG) is an immunomediated disorder which accounts for the most common cause of nephrotic syndrome (NS) following allogeneic hematopoietic stem cell transplantation (HSCT). OBJECTIVE AND METHODS: to provide an update on the issue by reviewing pertinent literature on the MEDLINE database. RESULTS: sixty-nine post allogenic HSCT patients (42 male) with MG were identified. The median age was 43 (5 to 68) years. Time interval from allogenic HSCT to MG diagnosis ranged from 3 to 134 months (median 17). Most MG patients had a history of acute (70%) or chronic (84%) graft versus host disease (GVHD). Corticosteroids and cyclosporine were the most common therapeutic agents used in this setting; alternative therapies, including rituximab, were given to a lower number of patients. Outcome data were available in 64 out of 69 MG patients; 38 (59%) and 18 (28%) patients achieved a complete and a partial response respectively, whereas treatment failure was recorded in the remaining 8 (13%). CONCLUSION: MG after allogenic HSCT appears to be associated with a sub clinical or overt cGVHD, which follows the discontinuation of immunosuppressive prophylaxis. Although a standard therapeutic approach has not been established, the application of available measures can induce favorable response in more than 80% of affected patients, but treatment-failure and progressive deterioration of the renal function may occur in about one fifth of cases.

The prevention of oral mucositis in patients with blood cancers: current concepts and emerging landscapes.

BACKGROUND: The prevention of oral mucositis (OM) in the management of hematological malignancies continues to represent an unmet clinical need. Addressing this issue has major clinical implications as OM can also greatly impair patient's quality of life. OBJECTIVES: To review currently available measures and investigational agents to prevent OM in hematological patients. METHODS: we searched for OM and related issues using Medline; the abstract books of the most important hematological and oncological meetings were also reviewed. RESULTS/CONCLUSIONS: Many agents targeting different mechanisms of mucosal damage have been applied in order to prevent OM; most of them have failed or its efficacy has not been fully demonstrated. Palifermin is the first pharmaceutical/biological agent approved for the prevention of OM; its use is currently restricted to patients who have received radiotherapy-containing conditioning regimens prior to autologous hematopoietic stem cell transplantation. No clear benefit by this agent has been demonstrated outside of this specific setting and its application should be limited to clinical trials. Other interventions, such as other growth factors and non mitogenic measures are under investigation or in development and their application in the hematological setting is expected in the short term.