Intrinsic OXPHOS limitations underlie cellular bioenergetics in leukemia.

Currently there is great interest in targeting mitochondrial oxidative phosphorylation (OXPHOS) in cancer. However, notwithstanding the targeting of mutant dehydrogenases, nearly all hopeful 'mito-therapeutics' cannot discriminate cancerous from non-cancerous OXPHOS and thus suffer from a limited therapeutic index. Using acute myeloid leukemia (AML) as a model, herein, we leveraged an in-house diagnostic biochemical workflow to identify 'actionable' bioenergetic vulnerabilities intrinsic to cancerous mitochondria. Consistent with prior reports, AML growth and proliferation was associated with a hyper-metabolic phenotype which included increases in basal and maximal respiration. However, despite having nearly 2-fold more mitochondria per cell, clonally expanding hematopoietic stem cells, leukemic blasts, as well as chemoresistant AML were all consistently hallmarked by intrinsic OXPHOS limitations. Remarkably, by performing experiments across a physiological span of ATP free energy, we provide direct evidence that leukemic mitochondria are particularly poised to consume ATP. Relevant to AML biology, acute restoration of oxidative ATP synthesis proved highly cytotoxic to leukemic blasts, suggesting that active OXPHOS repression supports aggressive disease dissemination in AML. Together, these findings argue against ATP being the primary output of leukemic mitochondria and provide proof-of-principle that restoring, rather than disrupting, OXPHOS may represent an untapped therapeutic avenue for combatting hematological malignancy and chemoresistance.

Day 14 Bone Marrow Evaluation During Acute Myeloid Leukemia Induction in a Real-world Canadian Cohort.

INTRODUCTION: The 2017 National Comprehensive Cancer Network guidelines for acute myeloid leukemia have recommended performing bone marrow (BM) aspiration and BM trephine biopsy (BMTB) 14 to 21 days after starting induction therapy (commonly referred to as "day 14 [D14] marrow"). Those who do not achieve a hypoplastic marrow, with cellularity < 20% and blasts < 5%, are recommended to undergo 2-cycle induction (2CI). We performed a retrospective analysis to determine the impact of D14 BM characteristics in predicting for remission, association with overall survival (OS), and the effect of 2CI according to the D14 BM results. PATIENTS AND METHODS: Patients aged 18 to 70 years undergoing induction therapy with standard "7 + 3" regimens were included. D14 cellularity was determined from BMTB samples and the blast percentage was assessed by morphology on BM aspiration and BMTB samples. The outcomes evaluated included the rates of complete remission (CR) and OS. RESULTS: A total of 486 patients with results from D14 BM evaluation were included in the present study. On multivariate analysis, cytogenetic risk and D14 blasts < 5% were predictive of CR/CR with incomplete count recovery (P < .001). Cytogenetic risk (P < .001), age < 60 years (P = .001), and D14 blasts < 5% (P = .045) predicted for OS. 2CI was performed in 131 patients (27%). Patients with hypocellular D14 BM but residual blasts (n = 106) underwent 2CI in 46% of cases, with improved remission rates (43.9% vs. 72.0%; P = .004) but no difference in OS. CONCLUSIONS: The results from D14 BM evaluations are predictive of subsequent remission and OS. Our findings did not show a survival benefit with D14 BM-driven 2CI.

Coffee and tea consumption during pregnancy and risk of childhood acute myeloid leukemia: A Childhood Leukemia International Consortium (CLIC) study.

BACKGROUND: Dietary habits during pregnancy have been inconsistently linked to childhood acute myeloid leukemia (AML), given the putative intrauterine onset of the disease as a result of triggering events during the critical period of fetal hematopoiesis. We investigated the potential association of maternal coffee and tea consumption during pregnancy with childhood AML risk, pooling primary data from eight case-control studies participating in the Childhood Leukemia International Consortium. METHODS: Information on coffee and/or tea consumption was available for 444 cases and 1255 age- and sex-matched controls, on coffee consumption for 318 cases and 971 controls and on tea consumption for 388 cases and 932 controls. Categories for cups of daily coffee/tea consumption were created in order to explore potential dose-response associations. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using logistic regression. RESULTS: Associations were found neither in the analysis on coffee or tea nor in the analysis on coffee only consumption (any versus no). A positive association with increasing coffee intake was observed (>1 cup per day; OR: 1.40, 95% CI: 1.03-1.92, increment of one cup per day; OR: 1.18, 95% CI: 1.01-1.39). No associations were observed with tea consumption. Interaction analyses showed non-significant associations between coffee/tea and smoking. Hyperdiploidy was inversely associated with tea consumption, with other cytogenetic markers having no association with coffee/tea. CONCLUSION: Given the widespread consumption of caffeinated beverages among pregnant women, our finding is of important public health relevance, suggesting adverse effects of maternal coffee consumption during pregnancy in the offspring.

Importance of Nutrition in the Treatment of Leukemia in Children and Adolescents.

BACKGROUND AND AIMS: Malnutrition has been identified as a prognostic factor in children and adolescents with leukemia. METHODS: A review of the data available on this topic has been carried out. RESULTS AND CONCLUSIONS: In children and adolescents (0-19 years of age), acute lymphoblastic leukemia (ALL) is the commonest form of cancer worldwide and malnutrition is prevalent in this age group, especially in low- and middle-income countries where most of these young people live. Obesity, measured by body mass index, is associated with poorer survival rates in children and adolescents with ALL and acute myelogenous leukemia in high-income countries. In contrast, undernutrition is linked to poorer survival rates among young people with leukemia in low- and middle-income countries.

Pediatric acute myeloid leukemia.

Pediatric acute myeloid leukemia (AML) is currently associated with survival rates as high as 70%. However, many events still occur, side effects are significant, and late effects occur and can even be life-threatening. Thus, the treatment of pediatric AML still needs further improvement. While most study groups agree on several principles of AML treatment, many unanswered questions and even controversies remain, which will be the topic of this review. Relapsed AML, the most frequent event in children, will also be discussed. The controversies justify future clinical studies. Fortunately, biotechnical developments provide novel treatment targets and targeted drugs, and will enable minimal residual disease-driven tailored therapy. Moreover, a wide range of new drugs is being developed. International collaboration is required to perform randomized, or even single-arm clinical studies, in this setting of subgroup-directed therapy, and fortunately is being accomplished. Therefore, optimism is justified and the treatment of pediatric AML will continue to improve.

Intra-abdominal Rhizopus microsporus infection successfully treated by combined aggressive surgical, antifungal, and iron chelating therapy.

SUMMARY: Zygomycetes are widely distributed in the environment as inhabitants of soil and decaying matter. On rare occasions, these organisms can cause invasive infections in immunocompromised hosts. As zygomycetes are resistant to most conventional antifungal agents, its infection is often fatal. We report 2 cases of unusual intra-abdominal Rhizopus microsporus infection in children with acute leukemia as a result of an unprecedented outbreak due to oral intake of contaminated allopurinol tablets and ready-to-eat food items. Among the 2 patients, one of them survived after aggressive combined surgical, antifungal (AmBisome, Caspofungin, and Posaconazole) and iron chelation therapy.

Lestaurtinib, a multitargeted tyrosine kinase inhibitor: from bench to bedside.

IMPORTANCE OF THE FIELD: Internal tandem duplication of the fms-like tyrosine kinase 3 (FLT3) gene (FLT3-ITD) is a common recurring mutation in acute myeloid leukemia (AML) with normal karyotype, and the presence of FLT3-ITD confers a poor prognosis on this large subgroup of AML patients. Since the discovery of lestaurtinib as a potent FLT3 inhibitor, in 1985, there has been considerable interest in the development of this agent (CEP-701, Cephalon, Frazer, PA, USA) for treatment of this population. AREAS COVERED IN THIS REVIEW: An extensive literature search was conducted that included published articles and abstracts on the preclinical and clinical development of this agent spanning the last decade. WHAT THE READER WILL GAIN: The review describes the historical development of this agent and reviews the available preclinical and clinical data on lestaurtinib and expands on potential future directions in development of this agent. TAKE HOME MESSAGE: Lestaurtinib is a multi targeted tyrosine kinase inhibitor which has been shown to potently inhibit FLT3 at nanomolar concentrations in preclinical studies, leading to its rapid development as a potential targeted agent for treatment of AML. Phase I studies have shown lestaturtinib to be an active agent particularly when used in combination with cytotoxic drugs. Currently, Phase II and Phase III studies are underway aiming to establish the future of this agent as a treatment option for patients with FLT3-ITD AML.

Camptothecin and khat (Catha edulis Forsk.) induced distinct cell death phenotypes involving modulation of c-FLIPL, Mcl-1, procaspase-8 and mitochondrial function in acute myeloid leukemia cell lines.

BACKGROUND: An organic extract of the recreational herb khat (Catha edulis Forsk.) triggers cell death in various leukemia cell lines in vitro. The chemotherapeutics camptothecin, a plant alkaloid topoisomerase I inhibitor, was tested side-by-side with khat in a panel of acute myeloid leukemia cell lines to elucidate mechanisms of toxicity. RESULTS: Khat had a profound effect on MOLM-13 cells inducing mitochondrial damage, chromatin margination and morphological features of autophagy. The effects of khat on mitochondrial ultrastructure in MOLM-13 correlated with strongly impaired routine respiration, an effect neither found in the khat-resistant MV-4-11 cells nor in camptothecin treated cells. Enforced expression of anti-apoptotic Bcl-2 protein provided protection against camptothecin-induced cell death and partly against khat toxicity. Khat-induced cell death in MOLM-13 cells included reduced levels of anti-apoptotic Mcl-1 protein, while both khat and camptothecin induced c-FLIPL cleavage and procaspase-8 activation. CONCLUSION: Khat activated a distinct cell death pathway in sensitive leukemic cells as compared to camptothecin, involving mitochondrial damage and morphological features of autophagy. This suggests that khat should be further explored in the search for novel experimental therapeutics.

Investigational drugs targeting FLT3 for leukemia.

FMS-like tyrosine kinase-3 (FLT3) is a member of the class III membrane receptor tyrosine kinase family and is important in survival, proliferation and differentiation of hematopoietic cells. FLT3 is mutated in approximately 30% of acute myelogenous leukemia patients. These mutations involve internal tandem duplications in the juxtamembrane domain of the receptor and tyrosine kinase point mutations in the activation loop. Over the past decade, due to the incidence and poor prognosis associated with FLT3, numerous agents have been developed to directly inhibit the activity of wild type and mutated FLT3. In this review, we focus on the preclinical data demonstrating in vitro activity, inhibition of downstream signaling pathways and potential synergy with traditional chemotherapeutic agents. Also, early clinical trial data specifically focusing on drug toxicity, clinical efficacy and future directions of FLT3-directed anticancer therapy are discussed.

The flow cytometric pattern of CD34, CD15 and CD13 expression in acute myeloblastic leukemia is highly characteristic of the presence of PML-RARalpha gene rearrangements.

BACKGROUND AND OBJECTIVE: Rapid identification of AML patients carrying the t(15;17) translocation for treatment decision-making is currently made on the basis of morphologic screening. However, the existence of both false positives and negatives highlights the need for more objective methods of screening AML cases and further molecular confirmation of the t(15;17) translocation. DESIGN AND METHODS: In the present study we analyzed a total of 111 AML cases in order to investigate whether immunophenotyping based on the assessment of multiple-stainings analyzed at flow cytometry could improve the sensitivity and specificity of morphologic identification of acute promyelocytic leukemia (APL) carrying the t(15;17) translocation. FISH analysis was used as a complementary technique for cases in which morphology and molecular biology yielded discrepant results. RESULTS: Concordant results between morphology and RT-PCR were found in 102/111 (91.8%) cases: 34 patients had M3/PML-RARalpha+ and 68 non-M3/PML-RARalpha- disease. Nine cases showed discrepants results. Multivariate analysis showed that the best combination of immunologic markers for discriminating between M3/PML-RARalpha+ and non-M3/PML-RARalpha- cases was that of the presence of heterogeneous expression of CD13, the existence of a single major blast cell population, and a characteristic CD34/CD15 phenotypic pattern (p<0.02). A score system based on these parameters was designed, and the 34 M3/PML-RARalpha+ cases showed a score of 3 (presence of the 3 phenotypic characteristics). In contrast, only 1 out of the 68 (1.3%) non-M3/PML-RARalpha- cases had this score, most o these latter cases (53/68, 78%) scoring either 0 or 1. Therefore, among these cases, immunophenotyping showed a sensitivity of 100% and a specificity of 99% for predicting PML/RARalpha gene rearrangements. Of the 9 cases in which morphology and molecular biology results were discrepant, four cases displayed M3 morphology without PML/RARalpha rearrangements by RT-PCR. In only one of these 4 cases did the immunophenotype score 3, this being the only FISH positive case. From the remaining five discrepant cases (non-M3 morphology while positive for PML/RARalpha) two cases had a phenotypic score of 3 and were FISH positive while the other three were negative by FISH. Upon repeating RT-PCR studies, two of these latter three cases became negative. INTERPRETATION AND CONCLUSIONS: Our results show that immunophenotyping may be of great value for quick screening of APL with PML/RARalpha rearrangements.

Pharmacologic and psychologic interventions for procedural pain.

OBJECTIVE: This study evaluated a combined pharmacologic and psychologic intervention (combined intervention, CI) relative to a pharmacologic-only (PO) intervention in reducing child distress during invasive procedures in childhood leukemia. Predictors of child distress included age, group (CI, PO), and procedural variables (medications and doses, technical difficulty, number of needles required). METHODOLOGY: This was a randomized, controlled prospective study that compared the PO (n = 45) and CI arms (n = 47), at 1, 6, and >12 months after diagnosis. A cross-sectional control group consisted of parents of 70 patients in first remission before the prospective study. Parent questionnaires, staff and parent ratings, and data on medications administered, technical difficulty of the procedure, and needle insertions were obtained for each procedure. This article reports on the final data point for the project (>12 months). RESULTS: Mothers and nurses reported lower levels of child distress in the CI than the PO group. The CI and PO groups showed lower levels of child and parent distress than the cross-sectional control group. Distress decreased throughout the time, and child age was inversely related to distress (younger children had more distress) regardless of group. Child distress was associated with staff perceptions of the technical difficulty of the procedure and with child age, but not with medications administered. CONCLUSIONS: The data showed that pharmacologic and psychologic interventions for procedural distress were effective in reducing child and parent distress and support integration of the two approaches. Younger children experienced more distress and warranted additional consideration. Staff perceptions of the technical difficulty of procedures were complex and potentially helpful in designing intervention protocols.

[Observation of cardiac function, hemorheology and lingual microcirculation of leukemic patients].

The cardiac function, hemorheology and the lingual microcirculation of the 62 leukemic patients were investigated. The results showed that their left cardiac function was markedly damaged, and the blood specific viscosity and hematocrit lowered, the time of RBC electrophoresis extended, the ESR and the K value of ESR equation was increased, which suggested the blood agglutination was enhanced. The lingual microcirculation obviously displayed reducing the number of papilla fungiformis, of intrapapillar plexus of microvessel as well as vessel loops, it also revealed the increasing of the abnormal form of microvessel and of the dilating vessel loops. The colour of the blood flow showed pink and dark red, the condition around vessel loops showed exudation and bleeding in several cases.

Nutritional behaviour and quality of life during oncological polychemotherapy: results of a prospective study on the efficacy of oral nutrition therapy in patients with acute leukaemia.

A total of 29 patients with acute leukaemia were prospectively randomized before starting cytostatic treatment to be nourished either with intensified oral nutrition (intervention group) or ad libitum nutritional intake during the whole tumour therapy (median 22 weeks). All received menus of free choice (daily offer of 1.0-2.0 g protein, 30-50 kcal kg-1 body weight (BW)). Beyond this, intervention patients received nutrition education, daily visits by the dietician and record of food intake, as well as a weekly assessment of subjective well-being (linear analogue self assessment 'LASA'). From the LASA items, the factors: 'malaise', 'psychological distress', 'therapy side-effects' were extracted by principal component analysis, and correlated to nutrient intake and nutritional status. At the end of antineoplastic induction therapy, after continuous hospitalization of 10 weeks (median), 31.3% of the controls had regained their initial nutritional status, and 68.8% of the intervention group. Mean daily energy intake was 23.2 kcal kg-1 BW during weeks with weight loss (constant weight: 30.9, weight gain: 39.3 kcal kg-1 BW). Nutritional behaviour correlated with subjective well-being, low intake with complaints of tumour treatment side effects and weight loss with malaise.

Nutritional requirements of human malignant (leukemic) cell lines: implications for adjuvant therapy.

Metabolic requirements of malignant cell lines derived from patients with chronic myelogenous and acute lymphoblastic leukemias were compared to those of proliferating normal cells (mitogen-stimulated human lymphocytes) and circulating blasts from acute myeloblastic and acute lymphoblastic leukemias. Requirements were judged by degree of amino acid (AA) utilization in short-term cultures and assessed by the effect of selective AA deprivation on cell growth. Cell growth was measured by DNA synthesis and growth rate analysis. Six AAs (serine, threonine, methionine, valine, phenylalanine, and lysine) were appreciably utilized (52-87%) by IM-9, CEM, MOLT-4, and K-562 cells, but little or no utilization of these or any other AAs were noted in HSB cells, in leukemic blasts, or in mitogen-stimulated normal lymphocytes in short-term culture. Omission of lysine from culture media greatly inhibited cell growth (DNA synthesis by 91%), and cell density (by 83%) of IM-9 cells. However, omission of lysine, valine, serine, threonine, methionine, or phenylalanine had less of an effect on CEM and MOLT-4 cell lines. These observations demonstrate that under the conditions used the IM-9 cell line is uniquely dependent on extracellular lysine levels in contrast to the other cell lines studied. This suggests that human malignancies other than acute lymphoblastic leukemia which exhibits an obligate dependence on extracellular asparagine might be manageable by enzymatic degradation in vivo or by dietary restriction of indispensable AAs.

Accelerated bone formation causing profound hypocalcemia in acute leukemia.

A patient with acute monocytic leukemia and fibrosis presented with severe hypocalcemia producing tetany, myocardial failure, and ventricular tachycardia with torsades de pointes configuration. Hypophosphatemia, hypomagnesemia, an elevated alkaline phosphatase level, and osteosclerosis were also present. Bone marrow biopsy samples showed fibrosis and thickened bony trabeculae lined with large osteoblasts. Tetracycline labeling showed an increased rate of calcification. Complete remission of the leukemia and fibrosis was achieved with a single 3-week course of low-dose cytarabine and hydroxyurea, with resolution of the hypocalcemia and hypophosphatemia. Calcitriol and etidronate disodium were also administered. The calculated left ventricular ejection fraction increased from 15% to 55% with correction of the hypocalcemia. The hypocalcemia and hypophosphatemia in this patient probably resulted from accelerated bone formation stimulated by the leukemic cells. The high dose of calcitriol that this patient received may have contributed to the remission of the leukemia.

Differentiation induction in myeloid leukemic cells.

Work based on immortalised leukemic cell lines indicates that the maturation arrest in leukemia can be reversible. Successful differentiation induction would mean restoring the link between proliferation and differentiation. Human cell lines such as the promyelocytic HL-60 and the monoblastic U-937 can be induced to mature by incubation with a wide variety of agents, e.g. phorbol diesters, retinoic acid and 1,25-dihydroxycholecalciferol. In addition, mitogen-stimulated lymphocytes and some T-lymphocyte lines produce a polypeptide called the differentiation-inducing factor (DIF), which mediates maturation of HL-60 into macrophage-like cells with resulting proliferation inhibition. DIF also displays a primary growth inhibitory effect on certain subclones of the cell lines as well as on fresh clonogenic cells from patients with acute myeloid leukemia and on normal granulocyte-macrophage progenitors. Our data indicate that there is more than one way to induce differentiation in leukemia but final common pathways may exist. Complementary, synergistic, maturation effects are seen between some agents, which may become of clinical utility.

Chemotherapeutic susceptibility of human bone marrow progenitor cells and human myelogenous leukemia cells (HL-60) in co-culture: preliminary report.

We determined the chemotherapeutic susceptibility of normal human granulocyte progenitor cells (CFU-C) and acute myelogenous leukemia cells (HL-60) in co-culture. Nucleated bone marrow cells and HL-60 cells were mixed in 0.3% agar containing McCoy's 5A medium, fetal bovine serum, human placenta-conditioned medium, and various concentrations of chemotherapeutic agents. They were incubated in 5% humidified CO2 at 37 degrees C for 8-10 days. CFU-C and HL-60 colonies were differentiated morphologically. The formation of CFU-C was progressively inhibited with the increasing number of HL-60 cells, whereas the presence or absence of bone marrow cells did not influence the number of HL-60 colonies. In separate culture, HL-60 cells were more sensitive to vincristine than were CFU-C cells. In co-culture, however, the CFU-C became more sensitive to vincristine than in the separate culture. Similarly, co-culture CFU-C were more sensitive to daunorubicin than in separate culture. These data indicate that HL-60 leukemic cells exert inhibitory effects on normal bone marrow CFU-C; in such an inhibited state, normal bone marrow is more susceptible to certain chemotherapeutic agents.

Cytotoxicity of thioether-lysophospholipids in leukemias and tumors of human origin.

Thioether-lysophospholipids inhibited the in vitro incorporation of [3H]thymidine into blasts of 8 leukemias, lymphocytes of 3 chronic lymphocytic leukemias, and cells of 12 different solid tumors of human origin. This effect correlated with trypan blue dye exclusion, which was used to assess cell damage. Scanning electron microscopy revealed severe membrane destruction after incubation with thioether-lysophospholipids. Cytostatic and cytotoxic effects of thioether-lysophospholipids were dependent on dosage and incubation time. Destruction of leukemic blasts was completed with greater than or equal to 5 micrograms/ml after an incubation of greater than or equal to 48 hr, but 10 to 20 micrograms/ml were necessary in solid tumors. Ester-linked 2-lysophosphatidylcholine was ineffective in the same dose range, which points to the requirement of the alkyl moiety in SN1 of the molecule for the antineoplastic properties of lysophospholipid analogues.

Induced maturation of the human promyelocytic leukemia cell line, HL-60, by 2-beta-D-ribofuranosylselenazole-4-carboxamide.

The new synthetic nucleoside analogue, 2-beta-D-ribofuranosylselenazole-4-carboxamide, was evaluated for its effects upon the growth and maturation of the human promyelocytic leukemia cell line, HL-60. At a concentration of greater than or equal to 1 nm, this agent was found both to decrease HL-60 cell proliferation and to cause the cells to acquire an ability to phagocytose opsonized yeast and to reduce nitroblue tetrazolium dye, functions characteristic of mature myeloid cells. In addition, this agent at similar concentrations caused a marked depression of intracellular guanosine nucleotide pools and a reduction in the incorporation of [14C] hypoxanthine into guanylates. These results suggested that the selenazole nucleoside caused an inhibition of inosinate monophosphate dehydrogenase, a key enzyme of guanylate biosynthesis. We therefore measured the activity of this enzyme indirectly by simultaneous-UV-radioactivity HPLC as well as by a direct radiometric method and demonstrated markedly reduced enzyme activities by both assays in drug treated cells. Dose response studies indicated that concentrations of drug which caused greater than 30% inhibition of IMP dehydrogenase activity induced greater than 50% maturation of the cells. These observations with this new nucleoside analogue provide further support for the concept that production of guanosine nucleotides and the activity of IMP dehydrogenase have a role in regulating the terminal maturation of myeloid cells.