Subtype Of Acute Myelogenous Leukemia Research Articles

Discovery of JNJ-74856665: A Novel Isoquinolinone DHODH Inhibitor for the Treatment of AML.

Acute myelogenous leukemia (AML), a heterogeneous disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway and preclinical findings demonstrated that DHODH is a metabolic vulnerability in AML as inhibitors can induce differentiation across multiple AML subtypes. As a result of virtual screening and structure-based drug design approaches, a novel series of isoquinolinone DHODH inhibitors was identified. Further lead optimization afforded JNJ-74856665 as an orally bioavailable, potent, and selective DHODH inhibitor with favorable physicochemical properties selected for clinical development in patients with AML and myelodysplastic syndromes (MDS).

Characteristics and complications of acute promyelocytic leukemia in children: an analysis of a national database

Acute promyelocytic leukemia (APL) is an uncommon subtype of acute myelogenous leukemia (AML) that was previously one of the most fatal forms of acute leukemia. With advances in diagnosis and treatment, APL has become one of the most curable myeloid leukemias. The major reason for treatment failure in APL is early death after initiation of treatment. We performed a retrospective cross-sectional analysis of the Healthcare Cost and Utilization Project 2016 and 2019 Kids’ Inpatient Database, with the diagnosis of APL or AML not in remission as defined by ICD-10-CM codes. We compared complications and outcomes associated with APL and AML (exclusive of APL) in hospitalized children in the U.S. and described yearly national incidence. The national incidence of APL was 2.2 cases per million children per year. Children with APL were more likely to have cardiopulmonary complications (OR 1.79; CI 1.20–2.67; p = 0.004), coagulation abnormalities or DIC (OR 7.75; CI 5.81–10.34; p < 0.001), pulmonary hemorrhage (OR 2.18; CI 1.49–3.17; p < 0.001), and intracranial hemorrhage (OR 10.82; CI 5.90–19.85; p < 0.001) and less likely to have infectious complications (OR 0.48; CI 0.34–0.67; p < 0.001) compared to children with AML. In-hospital mortality rates were similar in children with APL and AML (4.2% vs 2.6%; OR 1.62; CI 0.86–3.06; p = 0.13), while the median length of stay for children who died from APL was shorter compared to AML (2 (IQR: 1–7) versus 25 (IQR: 5–66) days; p < 0.05). Hemorrhagic complications occur more often, and infectious complications occur less often in hospitalized children with APL compared to AML.

ACUTE LEUKEMIA IN CHILDREN - AN INPATIENT DESCRIPTIVE STUDY

Acute leukemia in children causes unacceptably high morbidity and mortality in the affected patients in our population. This study was undertaken to create local data that could be used to better understand the characteristics of this disease in our set up, provide better management to the affected children and help decrease the high morbidity and mortality associated with this disease. To determine the frequency of the significant classes of acute leukemia, its subclasses and rate of remission in our study population. This study was conducted in Pediatric B Unit, Department of Pediatrics, Hayatabad Medical Complex, Peshawar, from 1st June 2017 to 31st December 2018. Using consecutive sampling technique, 90 cases were selected. All children who underwent bone marrow examination during the study period in this hospital were included in the study. Data on age, gender, indications, and major types and subclasses of leukemia was extracted from bone marrow record forms of these children and analyzed using SPSS version 20. Means and standard deviations were calculated for age and frequency and percentages for gender, types, and subtypes of leukemia and remission. 90 patients were included in this study. TLC was low in 33 (36.7%). Normal in 24 (26.6%) and raised in 33(36.7%). Acute lymphoblastic leukemia- ALL -L1 accounted for 80 (88.9%) of cases, while acute myelogenous leukemia for 10(11.1%). Acute myelogenous leukemia subtypes included AML-M5 in 5 (5.6%), AML-M2 and AML-M3 (2.2%) AML-M0 2 (2.2%), and AML-M2 1 (1.1%). 43 (47.8%) were newly diagnosed cases. 47 (52.2%) were previously diagnosed cases, of which 26 (55.3%) were in remission. Acute lymphoblastic leukemia ALL-L1 was the commonest type of leukemia with ALL-L1 subtype in all patients followed by acute myelogenous leukemia with AML-M5 the commonest subtype. Rate of response to therapy was 55.3%.

Impact of Qualified Dedicated APL Leukemia Specialist and Simplified Treatment Algorithm on Early Deaths in APL, a Monocenter Experience

Introduction: Acute Promyelocytic Leukemia (APL) is a highly curable subtype of Acute Myelogenous Leukemia (AML). Since the incorporation of all-trans-retinoic acid ATRA and arsenic trioxide (ATO) survival rates for APL are reported to be >90% in clinical trials. Despite this, there is a discrepancy in survival rates in general practice, mainly due to significant early deaths (ED). Some national registries report 20-30% of patients die during induction. Major factors contributing to ED are due to rarity of the disease, advanced age, underlying comorbidities, lack of written guidelines for induction, delay in diagnosis, and physician experience level. Herein, we report our observations on the incorporation of a simple treatment algorithm and management by leukemia/APL experts on ED. Methods: Retrospective monocenter analysis of ED in APL patients who underwent induction therapy from 4/2007 to 3/2022. All APL patients who underwent induction therapy were stratified into 2 treatment groups. Patients in group 1 treated from 4/2007-12/2009 and 1/2014- 6/2017 by a hematologist without an algorithm. Group 2 patients were treated from 1/2010-12/2013 and 7/2017-03/2022 by an APL leukemia specialist using a written algorithm. Induction rate mortality was compared between both groups. The algorithm was a two-page set of guidelines directed at preventing or treating the major causes of early death. Institutional review board approval was obtained for this review. Results: Between 4/2007 and 3/2022, n=62 patients were analyzed. A total of n=5 patients were excluded from the study; n=3 passed away or were transitioned to hospice care prior to starting any form of induction therapy and n=2 were Jehovah's Witnesses who refused all blood products. A total of n=57 patients were stratified into their respective groups. Across all groups (n=10, 17.5%) reported ED, median age 68 (range 20-81), (n=6, 60%) patients >60 years of age. Group 1 consisted of n=23 patients, median age 53 (range 20-78), (n=12, 52.2%) were male, and (n=9, 39.1%) were high risk (WBC > 10,000/mm3). Incidence of ED was (n=9, 39.1%) from disseminated intravascular coagulation DIC (n=3), differentiation syndrome DS (n=4), multi-organ failure (n=1), infection (n=1). ATRA was initiated at suspicion of APL diagnosis in (n=23, 100%) of patients. ATRA given alone in (n=1, 4.3%) patients, combined with ATO in (n=8, 34.8%), with chemotherapy in (n=19, 82.6%), and (n=5, 21.7%) with ATO/ATRA/chemotherapy. Group 2 consisted of n=34 patients, median age 51 (range 10-88), (n=14, 41.2%) were male, (n=10, 41.2%) were reported as high risk. There were (n=1, 2.9%) reported ED from DIC. ATRA was initiated at suspicion of APL diagnosis in (n=34, 100%) of patients. ATRA was given alone in (n=7, 20.6%) patients, combined with ATO in (n=17, 50%) and chemotherapy in (n=8, 23.5%). ATRA/ATO/chemotherapy was administered in (n=6, 17.6%). Conclusions: Results of this retrospective study showed that a simplified treatment algorithm and management by APL experts decreased early death rates from 39.1% to 2.9%. This data is promising and has important implications for both academic centers and community practices.

Realgar (As4S4), a traditional Chinese medicine, induces acute promyelocytic leukemia cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway in vitro.

Acute promyelocytic leukemia (APL) is a specific subtype of acute myelogenous leukemia (AML) characterized by the proliferation of abnormal promyelocytes. Realgar, a Chinese medicine containing arsenic, can be taken orally. Traditional Chinese medicine physicians have employed realgar to treat APL for over a thousand years. Therefore, realgar may be a promising candidate for the treatment of APL. Nevertheless, the underlying mechanism behind realgar therapy is largely unclear. The present study aimed to investigate the effect of realgar on cell death in the APL cell line (NB4) in vitro and to elucidate the underlying mechanism. In this study, after APL cells were treated with different concentrations of realgar, the cell survival rate, apoptotic assay, morphological changes, ATP levels and cell cycle arrest were assessed. The expression of Bcl-2, Bax, Cytochrome C (Cyt-C) and apoptosis-inducing factor (AIF) at the mRNA and protein levels were also measured by immunofluorescence, quantitative PCR (qPCR) and Western blotting. We found that realgar could significantly inhibit APL cell proliferation and cell death in a time- and dose-dependent manner. Realgar effectively decreased the ATP levels in APL cells. Realgar also induced APL cell cycle arrest at the S and G2/M phases. Following realgar treatment, the mRNA and protein levels of Bcl-2 were significantly downregulated, whereas the levels of Bax, Cyt-C, and AIF were significantly upregulated. In summary, realgar can induce APL cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway, suggesting that realgar may be an effective therapeutic for APL.

N-Heterocyclic 3-Pyridyl Carboxamide Inhibitors of DHODH for the Treatment of Acute Myelogenous Leukemia.

Acute myelogenous leukemia (AML), a disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway; however, small molecule DHODH inhibitors were recently shown to induce differentiation in multiple AML subtypes. Using virtual screening and structure-based drug design approaches, a new series of N-heterocyclic 3-pyridyl carboxamide DHODH inhibitors were discovered. Two lead compounds, 19 and 29, have potent biochemical and cellular DHODH activity, favorable physicochemical properties, and efficacy in a preclinical model of AML.

The HMGCR Inhibitor Fluvastatin Induces Apoptosis and Autophagy in Primary and Neoplastic Mast Cells

Abstract Mast cell hyperplasia is noted in many inflammatory and allergic diseases. Mast cells can also generate a subtype of acute myelogenous leukemia. Understanding and treating mast cell hyperplasia and leukemia could be beneficial to both inflammatory and malignant diseases. Recently the family of statin drugs, widely employed as HMG CoA Reductase (HMGCR) inhibitors to lower serum cholesterol, have been found to be immunosuppressive. However, statin effects on mast cell survival are not known. We report that fluvastatin induces apoptosis in mouse bone marrow-derived mast cells (BMMC) and mastocytoma cells. Apoptosis was suppressed in p53 KO and Bcl-2 Tg BMMC, consistent with detectable mitochondrial damage and Caspase-9 activation. Because apoptosis induction could be mimicked by suppressing geranylgeranyl transferase (GGT), and fluvastatin effects were reversed by adding the product of the GGT reaction, loss of GGT-mediated protein lipidation is the likely mechanism of cell death. In addition to apoptosis, fluvastatin also induced autophagy. Chemical blockade of autophagy enhanced fluvastatin-mediated apoptosis in primary mast cells, but reduced it in mastocytomas. These data suggest that the FDA-approved statin drugs may be useful in suppressing mast cell hyperplasia and leukemia.

ASXL2 regulates hematopoiesis in mice and its deficiency promotes myeloid expansion.

Chromosomal translocation t(8;21)(q22;q22) which leads to the generation of oncogenic RUNX1-RUNX1T1 (AML1-ETO) fusion is observed in approximately 10% of acute myelogenous leukemia (AML). To identify somatic mutations that co-operate with t(8;21)-driven leukemia, we performed whole and targeted exome sequencing of an Asian cohort at diagnosis and relapse. We identified high frequency of truncating alterations in ASXL2 along with recurrent mutations of KIT, TET2, MGA, FLT3, and DHX15 in this subtype of AML. To investigate in depth the role of ASXL2 in normal hematopoiesis, we utilized a mouse model of ASXL2 deficiency. Loss of ASXL2 caused progressive hematopoietic defects characterized by myeloid hyperplasia, splenomegaly, extramedullary hematopoiesis, and poor reconstitution ability in transplantation models. Parallel analyses of young and >1-year old Asxl2-deficient mice revealed age-dependent perturbations affecting, not only myeloid and erythroid differentiation, but also maturation of lymphoid cells. Overall, these findings establish a critical role for ASXL2 in maintaining steady state hematopoiesis, and provide insights into how its loss primes the expansion of myeloid cells.

Frontline treatment of AML in adults.

Despite recent progress in diagnosis and leukemogenesis based on genomic landscapes in acute myelogenous leukemia (AML), advances in AML treatment lag behind. Over the past four decades, combination chemotherapy with anthracycline and cytarabine remains the standard induction therapy. Subsequent post-remission consolidation therapy stratifies patients into favorable-risk, intermediate-risk, and unfavorable-risk groups to assign post-remission therapies based on cytogenetic abnormalities and molecular mutations. Allogeneic stem-cell transplant decreases the risk of AML recurrence compared to standard chemotherapy, but it also raises the risk of serious complications. Recent large collections of matched genomic and clinical data may assist in selecting the best individualized therapy for each AML patient. Emerging evidence indicates that molecularly targeted therapies such as FLT3 and IDH inhibitors may be effective in distinct AML subtypes, providing further rationale for a personalized medicine approach. An umbrella trial, such as "BEAT AML Master Trial," designed to offer novel targeted therapy to AML patients based on their genetic characteristics, will be launching worldwide in the near future.

The HMGCR inhibitor Fluvastatin induces apoptosis and autophagy in primary and neoplastic mast cells

Abstract Mast cells are best known for their role in inflammatory and allergic disease. Mast cell leukemia is a rare subtype of acute myelogenous leukemia, typically expressing the c-Kit mutation, D816V, that yields factor-independent growth. Understanding and treating mast cell hyperplasia and leukemia could be beneficial to both inflammatory and malignant diseases. Recently the family of statin drugs, widely employed as HMG CoA Reductase (HMGCR) inhibitors to lower serum cholesterol, have also been found to be immunosuppressive. However, statin effects on mast cell survival are not known. We report that Fluvastatin induces apoptosis in mouse bone marrow-derived mast cells (BMMC) and P815 mastocytoma cells in a dose- and time-dependent manner. Apoptosis was evidenced by DNA fragmentation and caspase activation. Importantly, the mastocytoma line P815, which expresses mutant c-Kit, was readily killed by Fluvastatin, with an IC50 of 5–10μM. P815 cell death was marked by c-Kit upregulation preceding mitochondrial membrane instability and Caspase-9 activation. Chemical blockade of autophagy reduced Fluvastatin-mediated apoptosis in primary mast cells, but augmented apoptosis in transformed mast cells. These data suggest that the HMGCR pathway may be targeted to suppress mast cell survival, and that its combination with autophagy inhibitors may be useful for treating mast cell neoplasias.

ASXL2 Is Recurrently Mutated in t(8;21) AML and Regulates Hematopoietic Development

Chromosomal translocation t(8;21) (q22;q22) leading to generation of oncogenic RUNX1-RUNX1T1 fusion is a cytogenetic abnormality observed in about 10% of acute myelogenous leukemia (AML). Studies in animal models and recent next generation sequencing approaches have suggested cooperativity of secondary genetic lesions with t(8;21) in inducing leukemogenesis. In this study, we used targeted and whole exome sequencing of 93 cases (including 30 with matched relapse samples) to profile the mutational landscape of t(8;21) AML at initial diagnosis and post-therapy relapse. We identified recurrent mutations of KIT, TET2, MGA, FLT3, NRAS, DHX15, ASXL1 and KMT2Dgenes in this subtype of AML. In addition, high frequency of truncating alterations in ASXL2 gene (19%) also occurred in our cohort. ASXL2 is a member of mammalian ASXL family involved in epigenetic regulation through recruitment of polycomb or trithorax complexes. Unlike its closely related homolog ASXL1, which is mutated in several hematological malignancies including AML, MDS, MPN and others; mutations of ASXL2 occur specifically in t(8;21) AML.We observed that lentiviral shRNA-mediated silencing of ASXL2 impaired in vitro differentiation of t(8;21) AML cell line, Kasumi-1, and enhanced its colony forming ability. Gene expression analysis uncovered dysregulated expression of several key hematopoiesis genes such as IKZF2, JAG1, TAL1 and ARID5B in ASXL2 knockdown Kasumi-1 cells. Further, to investigate implications of loss of ASXL2 in vivo, we examined hematopoiesis in Asxl2 deficient mice. We observed an age-dependent increase in white blood cell count in the peripheral blood of Asxl2 KO mice. Myeloid progenitors from Asxl2 deficient mice possessed higher re-plating ability and displayed altered differentiation potential in vitro. Flow cytometric analysis of >1 year old mice revealed increased proportion of Lin-Sca1+Kit+ (LSK) cells in the bone marrow of Asxl2 deficient mice, while the overall bone marrow cellularity was significantly reduced. In vivo 5-bromo-2’-deoxyuridine incorporation assay showed increased cycling of LSK cells in mice lacking Asxl2. Asxl2 deficiency also led to perturbed maturation of myeloid and erythroid precursors in the bone marrow, which resulted in altered proportions of mature myeloid populations in spleen and peripheral blood. Further, splenomegaly was observed in old ASXL2 KO mice and histological and flow cytometric examination of ASXL2 deficient spleens demonstrated increased extramedullary hematopoiesis and myeloproliferation compared with the wild-type controls. Surprisingly, loss of ASXL2 also led to impaired T cell development as indicated by severe block in maturation of CD4-CD8- double negative (DN) population in mice >1 year old. These findings established a critical role of Asxl2 in maintaining steady state hematopoiesis. To gain mechanistic insights into its role during hematopoietic differentiation, we investigated changes in histone marks and gene expression affected by loss of Asxl2. Whole transcriptome sequencing of LSK population revealed dysregulated expression of key myeloid-specific genes including Mpo, Ltf, Ngp Ctsg, Camp and Csf1rin cells lacking Asxl2 compared to wild-type control. Asxl2 deficiency also caused changes in histone modifications, specifically H3K27 trimethylation levels were decreased and H2AK119 ubiquitination levels were increased in Asxl2 KO bone marrow cells. Global changes in histone marks in control and Asxl2 deficient mice are being investigated using ChIP-Sequencing. Finally, to examine cooperativity between the loss of Asxl2 and RUNX1-RUNX1T1 in leukemogenesis, KO and wild-type fetal liver cells were transduced with retrovirus expressing AML1-ETO 9a oncogene and transplanted into irradiated recipient mice, the results of this ongoing study will be discussed.Overall, our sequencing studies have identified ASXL2 as a gene frequently altered in t(8;21) AML. Functional studies in mouse model reveal that loss of ASXL2 causes defects in hematopoietic differentiation and leads to myeloproliferation, suggesting an essential role of ASXL2 in normal and malignant hematopoiesis.*LH and NH contributed equally DisclosuresOgawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Disparities in the Upfront Use of Hematopoietic Stem Cell Transplant Among Patients with Acute Myeloid Leukemia

Background:The incidence for acute myelogenous leukemia (AML) is on the rise in the United States (US). Roughly 20,000 new AML patients are expected to occur in 2016 in the US (data from Surveillance Epidemiology and End Results Program). It is mainly a disease of older individuals with median age of 67 years. Unfortunately, most patients are not cured. Recent data suggest that early referral for hematopoietic stem cell transplant (HSCT) leads to more favorable outcomes, particularly those with non-good risk cytogenetics compared to alternate therapies. The objective of our study was to assess the use of upfront HSCT among AML patients with a focus on patients with non-good risk cytogenetics and determine disparity in its access based on sociodemographic and regional considerations.Methods:We analyzed data obtained from National Cancer Data Base (NCDB) Participant User Files for patients diagnosed with AML between 2004 and 2013 using the International Classification of Diseases for Oncology version 3 (ICD-O-3) codes 9840-9861, 9865-9874, 9891-9931. The data is collected prospectively by joint program of Commission on Cancer (CoC) and American Cancer Society. It covers nationwide oncology outcome from more than 1,500 CoC-accredited hospitals. Over 70% of all newly diagnosed cancer cases in the US are captured by NCDB. Since unique ICD-O-3 codes exist for the good-risk but not for intermediate- or poor-risk AML, we categorized the AML subtypes as good risk or non-good risk for analytic purposes. We considered patients with t(15;17), inv(16) and t(8;21) with corresponding ICD-O codes of 9871, 9896, and 9866, as good-risk patients. All others were considered non-good risk. Proportion of HSCT usage was estimated in various subgroups with 95% confidence intervals calculated using robust estimates of standard error.Results:We identified 80,087 patients who were diagnosed with AML between 2004 and 2013. Majority of these patients were Whites (85.9%), males (53.8%) and between the ages of 40 and 79 years (71.5%). The overall use of upfront HSCT increased consistently over time. This occurred predominantly among the non-good risk AML from a rate of 6.5% in 2004 to 12.5% in 2013 (Figure). Non-good risk patients who were Black, had Medicare insurance, with lower annual household income, and treated at facilities located in the East, South and Central parts of US ( AL, KY, MS, TN) had less access to HSCT (Table).Conclusion:The use of upfront HSCT in AML has almost doubled in the past decade, predominantly among those with non-good risk cytogenetics. Nevertheless, we found substantial disparity among sociodemographic and geographic subgroups. Future studies should try to understand and address how to bridge this gap. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Potential role for all-trans retinoic acid in nonpromyelocytic acute myeloid leukemia.

All-trans retinoic acid (ATRA) has been very successful in the subtype of acute myelogenous leukemia known as acute promyelocytic leukemia due to targeted reactivation of retinoic acid signaling. There has been great interest in applying this form of differentiation therapy to other cancers, and numerous clinical trials have been initiated. However, ATRA as monotherapy has thus far shown little benefit in nonacute promyelocytic leukemia acute myelogenous leukemia. Here, we review the literature on the use of ATRA in combination with chemotherapy, epigenetic modifying agents and targeted therapy, highlighting specific patient populations where the addition of ATRA to existing therapies may provide benefit. Furthermore, we discuss the impact of recent whole genome sequencing efforts in leading the design of rational combinatorial approaches.

Exome sequencing identifies highly recurrent somatic GATA2 and CEBPA mutations in acute erythroid leukemia.

Acute erythroid leukemia (AEL), characterized by a predominant erythroid proliferation, is a subtype of acute myelogenous leukemia. The genetic basis of AEL remains poorly defined. Through whole-exome sequencing, we identified high frequencies of mutations in CEBPA (32.7%), GATA2 (22.4%), NPM1 (15.5%), SETBP1 (12.1%) and U2AF1 (12.1%). Structure prediction analysis revealed that most of the GATA2 mutations were located at the DNA-binding N-terminal zinc-finger near the DNA-binding interface, suggesting that mutations could result in at least partial inactivation of GATA2 protein. On co-transfection of a GATA-responsive reporter construct together with plasmids expressing either GATA2 wild-type or GATA2 ZF1 mutants (P304H, L321P and R330X) in 293T cells, we found a reduced transcriptional activation in cells transfected with GATA2 mutants. To determine whether reduced GATA2 function is involved in leukemogenesis of AEL, we transfected 32D cells with GATA2 mutants and evaluated the impact of GATA2 mutations on erythroid differentiation. Our data revealed an increased expression of erythroid-related antigens Ter-119, β-globin and βh1-globin, as well as increased hemoglobin positivity in 32D cells transfected with GATA2 mutants compared with control cells. Our results suggest that the decline of GATA2 resulting from mutations contributes to the erythroid commitment, differentiation and the development of AEL.

Fluvastatin induces mast cell apoptosis and autophagy: effects on primary and transformed mast cells

Abstract Mast cells are best known for their role in inflammatory and allergic disease. Mast cell leukemia is a rare subtype of acute myelogenous leukemia, most of which express the c-Kit mutation, D816V, that yields factor-independent growth. Understanding and treating mast cell hyperplasia and leukemia could be beneficial to both inflammatory and malignant diseases. Recently the family of statin drugs, widely employed as HMG CoA Reductase inhibitors to lower serum cholesterol, have also been found to be immunosuppressive. However, statin effects on mast cell survival are not known. We report that Fluvastatin induces apoptosis in mouse bone marrow-derived mast cells (BMMC) and P815 mastocytoma cells in a dose- and time-dependent manner. Apoptosis was evidenced by DNA fragmentation and caspase activation. Importantly, the mastocytoma line P815, which expresses mutant c-Kit, was readily killed by Fluvastatin, with an IC50 of 5–10μM and apoptosis peaking at 72 hours. An up-regulation of the mutated c-Kit receptor was seen at 12 hours, followed by mitochondrial membrane instability and Caspase-9 activation within 24–28 hours. Finally, preliminary evidence of autophagy was detected during fluvastatin treatment. Interestingly, autophagy appeared to be cytoprotective in primary mast cells but cytotoxic in P815 mastocytoma cells. These preliminary data support the theory that statin family drugs induce mast cell autophagy and apoptosis, lending new therapeutic approaches for suppressing growth of both normal and transformed mast cells.

Fluvastatin elicits apoptosis in primary and transformed mast cells (INC6P.305)

Abstract Mast cell leukemia is a rare subtype of acute myelogenous leukemia. A majority of mast cell leukemias express the c-Kit mutation, D816V, which yields factor-independent growth. Understanding and treating mast cell hyperplasias and leukemias could be beneficial to both allergic and malignant diseases. Recently the family of statin drugs, widely employed as HMG CoA Reductase inhibitors to lower serum cholesterol, have also been found to be immunosuppressive. We report that Fluvastatin induces apoptosis in mouse bone marrow-derived mast cells (BMMC) in a dose-and time-dependent manner. Apoptosis was evidenced by DNA fragmentation and Caspase-3/7 activation. Interestingly, BMMC from the C57BL/6 background were responsive to Fluvastatin-mediated death, while those from 129/SvJ mice were completely resistant. Resistance was matched by a 2-fold induction of HMG CoA Reductase after Fluvastain treatment. Importantly, the mastocytoma line P815, which expresses mutant c-Kit, was readily killed by Fluvastatin, with an IC50 between 5-10µM, and maximum effects after 72 hours. Mitochondrial staining detected membrane disintegration after 48-72 hours, while cells tested positive for active Caspase-9 as early as 24-48 hours. These preliminary data support the theory that statin family drugs may be a new therapeutic approach for suppressing growth of both normal and transformed mast cells, and emphasize the contribution of genetic background to drug responsiveness.

Dual Promoter Usage as Regulatory Mechanism of let-7c Expression in Leukemic and Solid Tumors

Let-7c, an intronic microRNA (miRNA) embedded in the long non-coding gene LINC00478, can act as a tumor suppressor by targeting oncogenes. Previous studies indicated that in acute promyelocytic leukemia (APL), a subtype of acute myelogenous leukemia (AML) bearing the leukemia promoting PML/RARα fusion protein, let-7c expression seems to be controlled by the host gene promoter, in which canonical Retinoic Acid Responsive Elements (RAREs) are bound by PML/RARα in an all transretinoic acid (ATRA)-sensitive manner. Here, let-7c transcriptional regulation was further investigated and a novel intronic promoter upstream of the pre-miRNA was identified. This new promoter has transcriptional activity strongly indicating that at least two promoters need to be considered for let-7c transcription: the distal host gene and the proximal intronic promoter. Therefore, epigenetic modifying enzymes and histone acetylation and methylation status were analyzed on both let-7c promoters. It was demonstrated that ATRA treatment leads to let-7c upregulation inducing a more open chromatin conformation of the host gene promoter, with an enrichment of epigenetic marks that correlate with a more active transcriptional state. Conversely, the epigenetic marks on the intronic promoter are not significantly affected by ATRA treatment. Interestingly, in solid tumors such as prostate and lung adenocarcinoma it was found that both host and intronic promoters are functional. These data suggest that while the host gene promoter may control let-7c expression in AML, in a nonleukemic tumor context instead the intronic promoter contributes or preferentially regulates let-7c transcription. Alternative promoter usage represents a regulatory mechanism of let-7c expression in different tissues. Mol Cancer Res; 12(6); 878-89. ©2014 AACR.

Beneficial Effect Of Anticoagulants In The Management Of Patients With Acute Promyelocytic Leukemia (APL): Results Of a Multicenter, Retrospective Epidemiologic Study Of The Disseminated Intravascular Coagulation Patients In Japan

BackgroundAcute promyelocytic leukemia (APL) is an uncommon subtype of acute myelogenous leukemia (AML) with a high mortality rate, mostly attributable to intracranial hemorrhage caused by disseminated intravascular coagulation (DIC). Almost all patients with APL develop DIC. The introduction of all-trans retinoic acid (ATRA) into the induction chemotherapy regimen has revolutionized the treatment of individuals with APL, with nearly 90% of newly diagnosed APL patients achieving complete remission and over 70% of patients surviving longer than 5 years. However, population-based studies have shown that the early death-rate during induction chemotherapy remains extremely high with nearly 30% incidence and the most common cause of death is associated with hemorrhage. Thus, development of a novel treatment strategy to alleviate abnormal coagulation in APL patients is urgently required.In Japan, the conventional therapies for coagulopathy, such as low molecular heparin (LMWH), danaparoid sodium, and synthetic protease inhibitors were aggressively administered in addition to blood transfusions in DIC patients with APL. In 2008, a new anticoagulant, recombinant human soluble thrombomodulin (rTM) was approved for the treatment of DIC in Japan. Therefore, we accessed the difference of clinical safety and beneficial effects among these anticoagulant treatments in DIC patients with APL, by evaluating data on the multicenter, retrospective epidemiologic study of the DIC patients. Patients and MethodsPatients with DIC associated with newly diagnosed APL were recruited from January 2000 through December 2012 in 3 Japanese hematology centers. APL was defined according to FAB classification and was confirmed by the presence of t(15;17) and/or the PML-RARα fusion gene. DIC was evaluated by the diagnostic criteria of the Japanese Ministry of Health and Welfare. The treatment algorithm for newly diagnosed APL patients was generally based on the protocol proposed by the Japan Adult Leukemia Study Group (JALSG). The concomitant use of fresh frozen plasma, platelets, and/or other anticoagulant drugs were generally based on the recommendation of the JALSG protocol as follows: Platelet concentrate and fresh frozen plasma were transfused to maintain platelet counts ³30 × 109/L and plasma fibrinogen levels ³150 mg/dL, respectively.Early death (death within 30 day from APL diagnosis), severe hemorrhagic events, and improvement of coagulopathy were analyzed between rTM and other anticoagulants treatment. The time to disappearance of hemorrhagic symptoms between the two groups were analyzed by the Kaplan-Meier method and the log-rank test. ResultsDIC developed at diagnosis in 64 APL patients. 35 patients with DIC were treated with rTM (rTM group) and 29 patients with DIC were treated with LMWH, danaparoid sodium, and/or synthetic protease inhibitors (control group). There were no significant differences in age nor gender between rTM group and control group. Number of patients treated with ATRA alone and ATRA plus chemotherapy were 7 and 28 in the rTM group, and 10 and 19 in the control group. Early death rate for the rTM group was significantly lower than that for the control group (14% vs. 38%, P=0.043). Two patients developed intracranial hemorrhagic early death in the control group. On the other hand, no severe hemorrhagic event and mortality was noted in the rTM group. The time to disappearance of hemorrhagic symptoms was shorter for the rTM group than the control group (P=0.021). ConclusionsThese findings suggest that supportive care with rTM in combination with ATRA and chemotherapy ameliorates coagulopathy and reduces the risk of hemorrhagic early deaths in patients with APL than other anticoagulants. Disclosures:No relevant conflicts of interest to declare.

Pathogenesis of disseminated intravascular coagulation in patients with acute promyelocytic leukemia, and its treatment using recombinant human soluble thrombomodulin

Acute promyelocytic leukemia (APL) is an uncommon subtype of acute myelogenous leukemia characterized by the proliferation of blasts with distinct morphology, a specific balanced reciprocal translocation t(15;17), and life-threatening hemorrhage caused mainly by enhanced fibrinolytic-type disseminated intravascular coagulation (DIC). The introduction of all-trans retinoic acid (ATRA) into anthracycline-based induction chemotherapy regimens has dramatically improved overall survival of individuals with APL, although hemorrhage-related death during the early phase of therapy remains a serious problem. Moreover, population-based studies have shown that the incidence of early death during induction chemotherapy is nearly 30 %, and the most common cause of death is associated with hemorrhage. Thus, development of a novel treatment strategy to alleviate abnormal coagulation in APL patients is urgently required. Recombinant human soluble thrombomodulin (rTM) comprises the active extracellular domain of TM, and has been used for treatment of DIC since 2008 in Japan. Use of rTM in combination with remission induction chemotherapy, including ATRA, produces potent resolution of DIC without exacerbation of bleeding tendency in individuals with APL. This review article discusses the pathogenesis and features of DIC caused by APL, as well as the possible anticoagulant and anti-leukemic action of rTM in APL patients.

All-Trans Retinoic Acid–Induced Myositis and Retinoic Acid Syndrome in Microgranular-Variant Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is a subtype of acute myelogenous leukemia (AML). All-trans retinoic acid (ATRA) is an effective drug in the treatment of APL by promoting the terminal differentiation of leukemic cells into phenotypically mature myeloid cells. The microgranular-variant APL counts for a quarter of APL and is similar to monocytic origin leukemia. The microgranular-variant type has no effect on APL treatment. ATRA is related with some serious side effects such as Sweet’s syndrome and retinoic acid syndrome (RAS). Histologic characteristics of RAS are seen in capillary leakage and infiltration of organs by mature myeloid cells. ATRA-induced myositis is rarely described in adults and rare in children with APL. There have been increasing reports of ATRA-induced myositis, with its frequent association with RAS and Sweet’s syndrome. We report a patient with ATRA-induced myositis and RAS in microgranular variant APL and review the previously reported cases in the literature of ATRA-induced myositis. * APL : acute promyelocytic leukemia; ATRA : all-trans retinoic acid; RAS : retinoic acid syndrome; FISH : fluorescence in situ hybridization; MRI : magnetic resonance imaging; CK : creatine kinase; ICU : intensive care unit; CT : computed tomography; WBC : white blood cell