Jose E. Guimaraes

Targeting miR-21 Induces Autophagy and Chemosensitivity of Leukemia Cells

Overexpression of oncomiR-21 has been observed in most cancer types, such as leukemia. This miR has been implicated in a number of cellular processes, including chemoresistance, possibly by directly modulating the expression of several apoptotic related proteins. It was recently shown to directly target Bcl-2 mRNA and upregulate Bcl-2 protein expression. Nevertheless, the possible effect of miR-21 in autophagy has never been addressed. This study investigates the effects of targeting miR-21 with antimiRs on chronic myeloid leukemia cellular autophagy and on associated drug sensitivity. We observed that miR-21 downregulation decreased cellular viability and proliferation, although no changes to the normal cell cycle profile were observed. miR-21 downregulation also caused increased programmed cell death and a decrease in the expression levels of Bcl-2 protein, although PARP cleavage was not affected, indicating that apoptosis was not the relevant mechanism underlying the observed results. Treatment with antimiR-21 caused an increase in the autophagy related proteins Beclin-1, Vps34 and LC3-II. Accordingly, autophagic vacuoles were visualized both by monodansylcadaverine (MDC) and acridine orange (AO) staining and also by transmission electron microscopy (TEM). Additionally, miR-21 downregulation increased K562 and KYO-1 cellular sensitivity to etoposide or doxorubicin. This chemosensitivity was reverted by pre-treating cells with 3-MA, an autophagy inhibitor. Finally, serum starvation (an autophagy inducer) also increased sensitivity to these drugs, confirming that autophagy sensitized these cells to the effect of these drugs. To the best of our knowledge, this is the first description of autophagy induction via miR-21 targeting and its involvement in drug sensitivity.

Sequential Combination of Gemtuzumab Ozogamicin and Standard Chemotherapy in Older Patients With Newly Diagnosed Acute Myeloid Leukemia: Results of a Randomized Phase III Trial by the EORTC and GIMEMA Consortium (AML-17)

PURPOSE This randomized trial evaluated the efficacy and toxicity of sequential gemtuzumab ozogamicin (GO) and standard chemotherapy in older patients with newly diagnosed acute myeloid leukemia (AML). PATIENTS AND METHODS Patients (n = 472) age 61 to 75 years were randomly assigned to induction chemotherapy with mitoxantrone, cytarabine, and etoposide preceded, or not, by a course of GO (6 mg/m(2) on days 1 and 15). In remission, patients received two consolidation courses with or without GO (3 mg/m(2) on day 0). The primary end point was overall survival (OS). RESULTS The overall response rate was comparable between the two arms (GO, 45%; no GO, 49%), but induction and 60-day mortality rates were higher in the GO arm (17% v 12% and 22% v 18%, respectively). With median follow-up of 5.2 years, median OS was 7.1 months in the GO arm and 10 months in the no-GO arm (hazard ratio, 1.20; 95% CI, 0.99 to 1.45; P = .07). Other survival end points were similar in both arms. Grade 3 to 4 hematologic and liver toxicities were greater in the GO arm. Treatment with GO provided no benefit in any prognostic subgroup, with the possible exception of patients age < 70 years with secondary AML, but outcomes were significantly worse in the oldest age subgroup because of a higher risk of early mortality. CONCLUSION As used in this trial, the sequential combination of GO and standard chemotherapy provides no benefit for older patients with AML and is too toxic for those age ≥ 70 years.

Limited synergistic effect of antisense oligonucleotides against bcr-abl and transferrin receptor mRNA in leukemic cells in culture

The synergistic use of antisense oligonucleotides (ASOs) towards the bcr-abl and the transferrin receptor (TfR) mRNA was studied in a chronic myeloid leukemia (CML) cell line, aiming to improve the efficiency of individual ASO treatment. At 20 microM concentration, bcr-abl ASOs reduced cell growth by 40% and was specific for cells that have the translocation: there was a 34% reduction of BCR-ABL protein. The TfR ASO reduced cell growth by 20% and decreased TfR protein by 24%. The ASOs were more potent at reducing cell growth when used in combination (respectively, -20 and -17% than bcr-abl ASO and TfR ASO when used individually at the 10 microM concentration), thus we postulate that there is synergism of action. Cell cycle analysis also revealed that the sub-G1 peak was bigger in the synergistic treatment.

Simultaneous targeting of P-gp and XIAP with siRNAs increases sensitivity of P-gp overexpressing CML cells to imatinib.

Abstract It is accepted that cancer chemoresistance may be due to overexpression of antiapoptotic proteins or P‐gp. This study investigated the effect of downregulation of X‐chromosome‐linked inhibitor of apoptosis (XIAP) and of simultaneous downregulation of XIAP and P‐gp on sensitivity to imatinib. The K562 and K562Dox (P‐gp overexpressing) chronic myeloid leukemia cell lines were used and downregulation of target proteins was achieved with siRNAs. Targeting XIAP moderately enhanced sensitivity to imatinib in both cell lines. Simultaneous targeting of XIAP and P‐gp further enhanced sensitivity to imatinib in the resistant K562Dox cells. In conclusion, simultaneous targeting of P‐gp and XIAP increases sensitivity of P‐gp overexpressing chronic myeloid leukemia cells to imatinib.

Effect of miR-128 in DNA Damage of HL-60 Acute Myeloid Leukemia Cells

miR-128 has been associated with cancer, particularly with leukemia. In particular, this miR has been described, together with other miRs, to allow the discrimination between AML (acute myeloid leukemia) and ALL (acute lymphoblastic leukemia). In addition, miR-128 is included in miR signatures which not only allow characterizing a particular subtype of AML but are also associated with worse clinical outcome in a subgroup of patients with high-risk molecular features of AML. Nevertheless, all the published studies are based on data from expression arrays and no functional studies have been performed. Therefore, in order to further understand the role of miR-128 in AML cells and in their response to some chemotherapy, overexpression of miR-128 was achieved with miR-mimics in an AML cell line (HL-60). This resulted in decreased cellular viability and increased sensitization to both etoposide and doxorubicin. Overexpression of miR-128 increased programmed cell death but had no effect on cell cycle profile, 1 apoptosis or autophagy, as no alterations were observed in the protein levels of PARP, pro-caspase-3, Vps34, Beclin-1 or LC3-II. In addition, miR-128 overexpression increased the levels of DNA damage, as could be concluded by an increase in the comets tail intensity in the comet assay, an increase in the number of DNA repair foci stained with either γ-H2AX or 53BP1 proteins, and an increase in the levels of these two proteins (observed by Western blot). To the best of our knowledge, this is the first association of miR-128 with DNA damage in a leukemia context.

Invasive Coinfection with Aspergillus and Mucor in a Patient with Acute Myeloid Leukemia

The authors report the case of a 58-year-old female patient with a life-threatening invasive fungal disease caused by a coinfection of Aspergillus and Mucor species that occurred during induction and consolidation chemotherapy for an acute myeloid leukemia. The disease was successfully treated with an aggressive therapeutic approach, which consisted of liposomal amphotericin B in combination with surgical exploration without compromising the treatment of her underlying disease. The case demonstrates the difficulties associated with establishing a diagnosis as well as the need for close observation to identify these infections, which are often misdiagnosed and only suspected late during the course of the disease. Doubts about the reliability of the diagnostic tools lead to uncertainties with regard to the choice of first-line drugs as well the selection of the most appropriate therapeutic strategy in hematologic patients.

Mucor Infection: Difficult Diagnosis

Mucormycosis is a rare, highly aggressive and usually fatal infection, which affects immunocompromised patients. This case report describes a patient with acute promyelocytic leukemia who received antifungal therapy for a suspected pulmonary Aspergillus infection. Material from a lobectomy suggested that on histologic grounds the diagnosis had to be changed to mucormycosis. High suspicion of a Mucor infection favors early detection and timely appropriate antifungal therapy, which is crucial for the prognosis of these patients.

Impact of tandem autologous stem cell transplantation and response to transplant in the outcome of multiple myeloma

BackgroundMultiple Myeloma (MM) is the commonest indication for autologous stem cell transplantation (ASCT).MethodsWe retrospectively analysed data from 85 patients with MM submitted to ASCT in our centre from 2000 to 2010: 132 ASCT were realized, 80 of them as tandem.ResultsAfter induction, 17.6% were in complete remission (CR), 41.2% in very good partial remission (VGPR) and 41.2% in partial remission (PR). After transplant 44.7% were in CR, 15.3% in VGPR and 40% in PR. With 22 months (range – 3 to 117 months) of median follow-up, median overall survival (OS) was 43 months and progression-free survival (PFS) 22 months. At 5 years, OS was 45.3% (36.7-53.9%, 95%) and PFS 24.5% (18-31%, 95%). Patients with CR after ASCT had significantly longer PFS as compared to patients with PR (27 vs 7 months; p = 0.034) but not when compared to patients with VGPR (27 vs 19 months, p = 0.485). The tandem approach represented an advantage in OS and PFS when compared to only one ASCT (31 vs 19 months - p = 0.018, and 40 vs 31 - p = 0.04, respectively).ConclusionsOur results highlight the impact of response to transplant in patients PFS and tandem modality showed to carry better PFS and OS then the single transplant.

Bortezomib: Potential Key Role in the Treatment of Multiple Myeloma-Related Acquired Hemophilia A.

As previously reviewed in this journal, acquired hemophilia A (AHA) is a rare autoimmune disorder due to autoantibodies against factor VIII (FVIII).1–3 The autoantibodies against FVIII, commonly described as polyclonal and of immunoglobulin G (IgG) class, may inhibit the interaction of FVIII with FIXa, phospholipids, and von Willebrand factor.1 This interaction typically results in isolated prolongation of activated partial thromboplastin time (aPTT), limited correction in mixing studies with normal plasma, low levels of FVIII, and detection of a FVIII inhibitor.2 In 50 to 60% of cases, AHA is apparently idiopathic. The remaining cases are related to solid tumors, lymphoproliferative diseases, autoimmune disorders, viral infections, drug exposure, and pregnancy.3,4 The association between multiple myeloma (MM) and AHA is extremely rare. In a meta-analysis, from 30 patients with AHA and hematooncological diseases, only 4 had MM.5 This report describes a case ofMM-related AHA responsive to proteasome inhibitor (PI)-based therapy. To our knowledge, this is the second published clinical report showing the efficacy of PIs in the treatment of AHA6 and the first such report in a case of MM-related AHA. A male patient, 87 years old, was diagnosed with smolderingMM (sMM) IgG/kappa in February 2011:myeloma protein (M component) of 25 g/L, immunofixation positive for IgG/ kappa gammopathy, and plasmocytes accounting for 11% of nuclear cells in bonemarrow smear. Some 13months later, in April 2012, he developed anemia (hemoglobin [(Hb]: 8.9 g/dL) and progressed to symptomatic MM, Stage II-A in DurieSalmon Staging System and Stage II in International Staging System. He was initially treated with seven cycles of melphalan, prednisone, and thalidomide (MPT), achieving a minimal response with a reduction of 41% on the M component, from 22.2 to 13.2 g/dL. After 16 months with thalidomide maintenance therapy without progression, in July 2014, he appeared with cutaneous ecchymoses and hematomas, a tumefaction in the abdominal wall and an increase in diameter of the right thigh. The ultrasonography revealed two large hematomas: one (12 9 3.5 cm) in the left rectus abdominal muscle and another (19 12 11 cm) in the right thigh. The Hb decreased to 8.0 g/dL and an isolated prolonged aPTT of 73.6 seconds (normal: 24–36 seconds) was observed, with limited correctionwith 1:1mixture (40 seconds)with normal plasma (28 seconds). An incubated mix test was not performed. The FVIII activitywas 1.4 U/dL and FVIII inhibitor titer was determined to be 18.4 Bethesda Units (BU). Lupus anticoagulant and autoimmunity markers were negative. AHA was diagnosed and combined therapy with activated prothrombin complex concentrate (FEIBA) 50 IU/kg each for 8 hours (3,500 IU, three times/day) and cyclophosphamide 50 mg/day plus dexamethasone 20 mg/week was started. After 2 weeks, both hematomas and Hb became stable and the administration of FEIBA was suspended. After 1 month of immunosuppression, the aPTT lowered to 44.6 seconds, FVIII activity increased to 13U/dL, and FVIII inhibitor titer dropped to 8 BU (►Table 1). In October 2014, after 2 months of immunosuppression, the patient developed cellulitis on the right thigh. The ultrasonography revealed persistence of hematoma in the right thigh (14 6 cm) and Hb dropped to 7.9 g/dL. The aPTT (41 Letter to the Editor

Pyoderma gangrenosum and erythema elevatum diutinum associated with a high-risk myelodysplastic syndrome: case report

The authors report a case of Pyoderma gangrenosum (PG) with some clinical features mimicking PAPA (Pyogenic Arthritis, Pyoderma gangrenosum and Acne) syndrome, indicating a myelodysplastic syndrome (MDS). During the course of MDS, lesions of another neutrophilic dermatosis, erythema elevatum diutinum (EED), emerged. PG and EED are uncommon neutrophilic dermatoses of unknown etiology and their pathogenesis is poorly understood. In a recent review, up to 50% of PG cases are associated with an underlying systemic disease such as inflammatory bowel disease, rheumatoid arthritis or hematologic disorder.1 Among them, acute myeloid leukemia (AML) is found in 7% of the cases.1,2 Furthermore, EED has been associated with these conditions.2 On the other hand, extracutaneous manifestations of neutrophilic dermatosis are rare. Just a few reports describe PG with involvement of the lungs, joints, liver, spleen, bone or blood vessels.3