Hedgehog (Hh) signaling pathway is activated in diffuse large B-cell lymphoma (DLBCL). Genetic abnormalities that explain activation of Hh signaling in DLBCL are unknown. We investigate the presence of amplifications of Hh genes that might result in activation of this pathway in DLBCL. Our data showed few extra copies of GLI1 and SMO due to chromosomal aneuploidies in a subset of DLBCL cell lines. We also showed that pharmacologic inhibition of PI3K/AKT and NF-KB pathways resulted in decreased expression of GLI1 and Hh ligands. In conclusion, our data support the hypothesis that aberrant activation of Hh signaling in DLBCL mainly results from integration of deregulated oncogenic signaling inputs converging into Hh signaling.
Gene copy number; Hedgehog pathway; GLI; diffuse large B-cell lymphoma
The CBA/H mouse model of radiation-induced acute myeloid leukaemia (rAML) has been studied for decades to bring to light the molecular mechanisms associated with multistage carcinogenesis. A specific interstitial deletion of chromosome 2 found in a high proportion of rAML is recognised as the initiating event. The deletion leads to the loss of Sfpi, a gene essential for haematopoietic development. Its product, the transcription factor PU.1 acts as a tumour suppressor in this model. Although the deletion can be detected early following ionising radiation exposure by cytogenetic techniques, precise characterisation of the haematopoietic cells carrying the deletion and the study of their fate in vivo cannot be achieved. Here, using a genetically engineered C57BL/6 mouse model expressing the GFP fluorescent molecule under the control of the Sfpi1 promoter, which we have bred onto the rAML-susceptible CBA/H strain, we demonstrate that GFP expression did not interfere with X-ray induced leukaemia incidence and that GFP fluorescence in live leukaemic cells is a surrogate marker of radiation-induced chromosome 2 deletions with or without point mutations on the remaining allele of the Sfpi1 gene. This study presents the first experimental evidence for the detection of this leukaemia initiating event in live leukemic cells.
Radiation; Live cells; Chromosome deletion; Sfpi1/PU.1; Myeloid leukaemia; Mouse model
In a phase II trial, 16 patients with relapsed refractory multiple myeloma received temsirolimus 25 mg IV weekly until progression. One partial response and 5 minor responses were observed for a total response rate of 38%. The median time to progression was 138 days. Grade 3–4 toxicity included fatigue (n=3), neutropenia (n=2), thrombocytopenia (n=2), interstitial pneumonitis (n=1), stomatitis (n=1) and diarrhea (n=1). Clinical activity was associated with a higher area under the curve (AUC) and maximal reduction in phosphorylated p70S6K and 4EBP1 in peripheral blood mononuclear cells. At the dose and schedule used, temsirolimus had low single agent activity. Investigation of alternate dosing schedules and use in combinations is indicated.
Multiple myeloma; mTOR; temsirolimus; pharmacokinetics
Patients with acute myeloid leukemia (AML) receiving induction chemotherapy (IC) were enrolled in a supervised exercise intervention to determine safety, feasibility, and efficacy. Physical fitness measures, quality of life (QOL) and fatigue were assessed using standardized measures at baseline, post-induction, and post first consolidation. Retention was excellent, the intervention was safe, and efficacy estimates suggested benefits in physical fitness and QOL outcomes. Exercise is a safe, promising intervention for improving fitness and QOL in this patient population. These results provide a foundation for a randomized trial to better understand the impact of exercise during IC on clinically important outcomes.
Acute myeloid leukemia; Chemotherapy; Exercise; Pilot study; Quality of life; Cancer-related fatigue; Elderly; Physical fitness
We report a consecutive series of 59 patients with MDS who underwent reduced-intensity hematopoietic stem cell transplantation (RI-HSCT) with fludarabine/melphalan conditioning and tacrolimus/sirolimus-based GVHD prophylaxis. Two-year OS, EFS, and relapse incidences were 75.1%, 65.2%, and 20.9%, respectively. The cumulative incidence of non-relapse mortality at 100 days, 1 year, and 2 years was 3.4%, 8.5%, and 10.5%, respectively. The incidence of grade II-IV acute GVHD was 35.4%; grade III-IV was 18.6%. Forty of 55 evaluable patients developed chronic GVHD, 35 extensive grade. This RI-HSCT protocol produces encouraging outcomes in MDS patients, and tacrolimus/sirolimus-based GVHD prophylaxis may contribute to that promising result.
myelodysplastic syndrome; sirolimus; tacrolimus; allogeneic hematopoietic cell transplantation
The decision to re-induce patients with acute myeloid leukemia (AML) based on results of the day 14 bone marrow (BM) biopsy is variable and lacks evidence based data. The aim of our review was to evaluate the accuracy of a day 14 BM biopsy in determining the need for re-induction chemotherapy.
Seventy-four patients with newly diagnosed de novo AML treated with induction chemotherapy were retrospectively reviewed for the purpose of evaluating treatment decisions and outcomes based on their day 14 BM biopsy. Response to therapy in this analysis was based on morphology alone.
Of the 74 patients undergoing standard induction, 45 patients (61%) had no evidence of leukemia on their day 14 BM biopsy. Eighteen patients (24%) had definitive residual disease (RD), and 11 patient’s (15%) were classified as indeterminate response (IR). Fifteen patients with RD and one with IR underwent re-induction chemotherapy. However, thirteen patients (3 RD and 10 IR) were observed until count recovery without any re-induction therapy. Eleven of these 13 patients who were observed eventually attained a morphologic complete remission (CR), including two patients with RD.
A day 14 BM biopsy may have suboptimal sensitivity for the detection of residual leukemia. Some patients with an IR on day 14 may not require re-induction chemotherapy, but instead, may benefit from careful observation until count recovery to avoid the mortality and morbidity associated with re-induction chemotherapy.
AML; D14 BM biopsy; Re-induction
We previously demonstrated upregulation of c-myc, survivin, and cyclin D1 in CD34+ bone marrow mononuclear cells (BMMNCs) of patients with trisomy 8 and monosomy 7 myelodysplastic syndromes (MDS). “Knockdown” of cyclin D1 by RNA interference decreased trisomy 8 cell growth, suggesting that this might be a therapeutic target in MDS.
We performed preclinical studies using BMMNCs from patients with MDS and AML to examine the effects of the styryl sulfone ON 01910.Na on cyclin D1 accumulation, aneuploidy, and CD34+ blast percentage. We next treated twelve patients with higher risk MDS and two trisomy 8 AML patients with ON01910.Na on a phase I clinical protocol (NCT00533416).
ON 01910.Na inhibited cyclin D1 expression, and was selectively toxic to trisomy 8 cells in vitro. Flow cytometry studies demonstrated increased mature CD15+ myeloid cells and decreased CD34+ blasts. Three patients treated with ON01910.Na on a clinical had decreased bone marrow blasts by ≥50%, and three patients had hematologic improvements, one of which was sustained for 33 months. Patients with hematologic responses to ON 01910.Na had decreased cyclin D1 expression in their CD34+ cells.
The preclinical results and responses of patients on a clinical trial warrant further investigation of ON 01910.Na as a potential novel targeted therapy for higher risk MDS patients.
MDS; Treatment; ON 01910.Na; Cyclin D1
Despite improvements in disease management, multiple myeloma (MM) remains incurable. Conventional treatment methods are unsatisfactory, leading to a pattern of regression and remission, and ultimately failure. This pattern suggests that one of the possible strategies for improving outcomes is continuous therapy to maintain suppression of the surviving tumor cells. Optimal management of MM requires potent agents and modalities with direct tumoricidal activity, which can also provide continuous suppression of the residual tumor to prevent disease relapse. Immunomodulatory agents exert immunomodulatory and tumoricidal effects, and cause disruption of stromal cell support from the bone marrow microenvironment. Therefore continuous therapy with immumomodulatory agents may be able to provide both tumor reduction and tumor suppression, enabling physicians to consider the possibility of incorporating continuous therapy into the treatment paradigm of patients with MM.
Multiple myeloma; Cell biology; Bone marrow microenvironment; Mode of action; Immunomodulatory drugs; Proteosome inhibitors
Caspase function is known to be essential for cell death by apoptosis, but it is now increasingly recognized that these proteases also play important roles in other cellular events. Here we report for the first time that inhibition of cellular caspase activity can induce differentiation of AML blasts, and can enhance vitamin D-induced cell differentiation of these cells. This was studied in blasts obtained from nine patients with AML and one patient with CML by ex vivo culture in the presence of Q-VD-OPh (QVD), a pan caspase inhibitor. Cell differentiation was manifested by the expression of markers of monocytic differentiation CD11b and CD14. Differentiation induced by 1α,25-dihydroxyvitamin D (1,25D) or its analogs PRI-1906 and PRI-2191 was enhanced by QVD to a varying degree, depending on the subtype of the leukemia. QVD and 1,25D-induced differentiation was accompanied by increased signaling by Hematopoietic Progenitor Kinase 1(HPK1), and the expression of transcription factors known to be involved in monocytic differentiation was increased. Although the magnitude and nature of these changes were not invariable, it is clear that caspase inhibitors warrant attention as components of differentiation therapy of leukemia, perhaps in combination with derivatives of vitamin D.
Caspases; AML; CML; vitamin D; HPK1 pathway; Q-VD-OPh
Somatic CBL mutations have been reported in a variety of myeloid neoplasms but are rare in acute lymphoblastic leukemia (ALL). We analyzed 77 samples from hematologic malignancies, identifying a somatic mutation in CBL (p.C381R) in one patient with T-ALL that was associated with a uniparental disomy at the CBL locus and a germline heterozygous mutation in one patient with JMML. Two NOTCH1 mutations and homozygous deletions in LEF1 and CDKN2A were identified in T-ALL cells. The activation of the RAS pathway was enhanced, and activation of the NOTCH1 pathway was inhibited in NIH 3T3 cells that expressed p.C381R. This study appears to be the first to identify a CBL mutation in T-ALL.
CBL; acute lymphoblastic leukemia; Noonan syndrome; RAS; NOTCH
The inhibitors of apoptosis (IAP) are important regulators of apoptosis. However, little is known about the capacity of Smac mimetics (IAP inhibitor) to overcome virally-associated-lymphoma’s (VAL) resistance to apoptosis. Here, we explored the pro-apoptotic effect of a novel Smac mimetic, RMT5265.2HCL (RMT) in VAL cells. RMT improved the sensitivity to apoptosis in EBV- and to some extend in HTLV-1- but not in HHV-8-VAL. Furthermore, we identified that RMT promotes caspase 3 and 9 cleavage by inhibiting XIAP and inducing the mitochondrial efflux of Smac and cytochrome C. This investigation further support exploring the use of Smac inhibitors in VAL.
XIAP; EBV; HTLV-1; HHV-8; lymphomas; Smac mimetics
In this study, we show that conditioned media (CM) generated from bone marrow (BM)-derived mesenchymal stromal cells lead to BCR-ABL independent STAT3 activation. Activation of STAT3 is important not only for survival of CML cells but also for its protection against Nilotinib (NI), within the BM microenvironment. Reducing the expression of both JAK2 and TYK2 or utilizing a pan-JAK inhibitor blocked CM-mediated STAT3 activation and sensitized CML cells to NI-mediated cell death. Finally, we demonstrate that in patient-derived primitive leukemic cells, co-cultured with BM stromal cells, inhibition of BCR-ABL and JAK activity was a successful strategy to potentiate their elimination.
STAT3; Conditioned media; Bone marrow microenvironment; Nilotinib; JAK; INC424
Vitamin D deficiency has been linked with increased cancer risk, and vitamin D has been shown to be cytotoxic to some cancer cells in vitro. In the present study we evaluated whether vitamin D would have antiproliferative or cytotoxic effects on human pre-B acute lymphoblastic leukemia cells. Contrary to our hypotheses, calcitriol, the active form of vitamin D, had no effect on leukemia cell proliferation. Calcitriol actually had a modest effect to impair dexamethasone cytotoxicity and induction of apoptosis. Further studies are needed to evaluate the effects of vitamin D on leukemia cells in vivo.
Vitamin D; calcitriol; acute lymphoblastic leukemia; dexamethasone
Some oncolytic viruses, such as myxoma virus (MYXV), can selectively target malignant hematopoietic cells, while sparing normal hematopoietic cells. This capacity for discrimination creates an opportunity to use oncolytic viruses as ex vivo purging agents of autologous hematopoietic cell grafts in patients with hematologic malignancies. However, the mechanisms by which oncolytic viruses select malignant hematopoietic cells are poorly understood. In this study, we investigated how MYXV specifically targets human AML cells. MYXV prevented chloroma formation and bone marrow engraftment of two human AML cell lines, KG-1 and THP-1. The reduction in human leukemia engraftment after ex vivo MYXV treatment was dose-dependent and required a minimum MOI of 3. Both AML cell lines demonstrated MYXV binding to leukemia cell membranes following co-incubation: however, evidence of productive MYXV infection was observed only in THP-1 cells. This observation, that KG-1 can be targeted in vivo even in the absence of in vitro permissive viral infection, contrasts with the current understanding of oncolytic virotherapy, which assumes that virus infection and productive replication is a requirement. Preventing MYXV binding to AML cells with heparin abrogated the purging capacity of MYXV, indicating that binding of infectious virus particles is a necessary step for effective viral oncolysis. Our results challenge the current dogma of oncolytic virotherapy and show that in vitro permissiveness to an oncolytic virus is not necessarily an accurate predictor of oncolytic potency in vivo.
leukemia; oncolytic virotherapy; bone marrow; hematopoietic stem cell; animal models
Large granular lymphocyte (LGL) leukemia characterized by clonal expansion of antigen-activated cytotoxic T cells (CTL). Patients frequently exhibit seroreactivity against a human T-cell leukemia virus (HTLV) epitope, BA21. Aplastic anemia, paroxysmal nocturnal hemoglobinuria and myelodysplastic syndrome are bone marrow failure diseases that can also be associated with similar aberrant CTL activation (LGL-BMF). We identified a BA21 peptide that was specifically reactive with LGL leukemia sera and found significantly elevated antibody reactivity against the same peptide in LGL-BMF sera. This finding of shared seroreactivity in LGL-BMF conditions and LGL leukemia suggests that these diseases might share a common pathogenesis.
LGL leukemia; aplastic anemia; myelodysplastic syndrome; paroxysmal nocturnal hemoglobinuria; HTLV antibody
This study compares outcomes of low-intensity versus standard-intensity induction strategies for older patients with acute myeloid leukemia at the Weill Cornell Leukemia Program. From 1999 to 2009, 298 adults ≥60 years with AML underwent induction chemotherapy with low-intensity and standard-intensity regimens, based on physician and patient preferences and investigational protocol availability. Overall, 33% of the cohort achieved complete remission with initial treatment, 23% with low-intensity induction and 53% with standard-intensity induction (P < 0.0001). The median overall survival was 6.5 months and there was no significant difference in overall survival between patients initially treated with a low-intensity regimen compared to those receiving standard-intensity induction. There were no differences in 30- or 60-day mortality between the two groups.
Leukemia; Acute; Myeloid; Drug therapy; Clinical trials; Aged; Remission induction
In a series of 105 patients with polycythemia vera, we retrospectively determined whether the JAK2V617F mutation correlated with severity of disease phenotype. Higher JAK2V617F allele burden correlated with more advanced myelofibrosis, greater splenomegaly, and higher white blood cell count, but not with age, gender, hematocrit level, or frequency of phlebotomy prior to cytoreductive therapy. Although a subgroup at increased risk for thrombosis was not clearly defined, there was a suggestion that frequency of thrombosis increased as the JAK2V617F allele burden increased. The JAK2V617F allele burden did not change significantly in treated patients with serial JAK2 analyses.
Myeloproliferative disorders; Treatment of polycythemia vera; JAK2V617F mutation; Allele burden; Splenomegaly; Myelofibrosis
Effects of the HDAC inhibitor LBH-589 (panobinostat) on fludarabine lethality toward acute myeloid leukemia (AML) cells were examined in vitro and in vivo. LBH-589 pretreatment sensitized U937, HL-60, and primary leukemia cells to fludarabine while blocking NF-κB activation accompanied by XIAP down-regulation and JNK activation. Pharmacologic or genetic JNK inhibition significantly attenuated LBH-589/fludarabine lethality, whereas XIAP over-expression diminished JNK activation and apoptosis. Combined in vivo treatment abrogated leukemia growth in a U937 xenograft murine model and substantially increased animal survival. These studies highlight the interplay between NF-κB activation, XIAP down-regulation, and JNK activation in anti-leukemic synergism between fludarabine and LBH-589.
AML; histone deacetylase inhibitor; fludarabine
Older patients with AML have a worse outcome compared to young patients. To study for potential contributors to their poor prognosis, we compared two NK-AML cohorts, young (< 60 years old) and old (> 60 years old), via high density SNP array analysis. Older patients had more genomic changes (1.83±0.23 vs. 1.16±0.2, p=0.037) and a trend for a higher number of copy number neutral loss of heterozygosity (0.5±0.2 vs. 0.24±0.08, p=0.088) compared to young patients. We speculate that complex genomic changes in NK-AML may be a sign of an increase in genomic instability and an indicator of a worse prognosis.
AML; Normal karyotype; SNP array; Old age
In a Phase I/II clinical trial, 13 higher risk red blood cell-dependent myelodysplastic syndrome (MDS) patients unresponsive to hypomethylating therapy were treated with the multikinase inhibitor ON01910.Na. Responses occurred in all morphologic, prognostic risk and cytogenetic subgroups, including four patients with marrow complete responses among eight with stable disease, associated with good drug tolerance. In a subset of patients, a novel nanoscale immunoassay showed substantially decreased AKT2 phosphorylation in CD34+ marrow cells from patients responding to therapy but not those who progressed on therapy. These data demonstrate encouraging efficacy and drug tolerance with ON01910.Na treatment of higher risk MDS patients.
MDS; Treatment; ON01910.Na; rigosertib; nanoimmunoassay; AKT signaling pathway
Lack of suitable mouse models for central nervous system (CNS)-associated leukemias has hindered mechanism-guided development of therapeutics. By transplanting retrovirus-transformed mouse erythroleukemia cells into syngeneic mice, we developed a new animal model of meningeal leukemia associated with rapid paralysis. Necropsy revealed massive proliferation of the leukemic cells in the bone marrow (BM) followed by pathological angiogenesis and invasion of the leukemic cells into the meninges of the CNS. Further analysis demonstrated that the erythroleukemia cells secreted high levels of VEGF and preferentially adhered in vitro to fibronectin. This unique animal model for meningeal leukemia should facilitate studies of engraftment and proliferation of leukemic cells in the BM and their invasion of the CNS as well as pre-clinical evaluation of experimental therapeutics for CNS-associated leukemias.
Animal Model; Meningeal leukemia; Murine erythroleukemia cells; Cell adhesion Angiogenesis; PU.1; Friend SFFV
Activation of the mTOR pathway subsequent to phosphatase and tensin homolog (PTEN) mutation may be associated with glucocorticoid (GC) resistance in acute lymphoblastic leukemia (ALL). The combination activity of rapamycin and dexamethasone in cell lines and xenograft models of ALL was determined. Compared with either drug alone, dexamethasone + rapamycin showed significantly greater apoptosis and cell cycle arrest in some cell lines, which was more frequently seen in T-lineage cell lines with PTEN mutation. The combination significantly extended the event-free survival of mice carrying PTEN mutated xenografts. Our data suggest that PI3K/mTOR pathway inhibitors could benefit patients with PTEN mutated T-ALL.
dexamethasone; rapamycin; acute lymphoblastic leukemia
Aberrations in IL-3, GM-CSF and G-CSF induced signaling are frequently reported in acute myeloid leukemia (AML). Herein, we utilized a unique human myeloid leukemic cell line, AML-193, which responds to all three cytokines to analyze the regulation at microRNA level. Using real-time PCR-based miRNA expression profiling, we investigated miRNA signatures regulated by IL-3, GM-CSF and G-CSF for n=704 miRNAs. We discovered that in addition to regulating specific miRNAs, these cytokines also regulate common set of miRNAs, which includes miR-590-5p, miR-219-5p, miR-15b and miR-628-5p. Taken together, we have identified novel candidate miRNAs that may be instructive during leukemic and normal hematopoiesis.
AML-193; AML; miRNA; IL-3; GM-CSF; G-CSF
BCR-ABL1 kinase-positive leukemia cells accumulate high numbers of DNA double-strand breaks (DSBs) induced by the reactive oxygen species (ROS) or cytotoxic agents. To repair these lesions and prevent apoptosis BCR-ABL1 kinase stimulates the efficiency of DSB repair in leukemia cells. Histone acetylation-dependent chromatin re-modeling plays a crucial role in this process. Here we report that leukemia cells expressing BCR-ABL1 kinase displayed an enhanced histone acetylase activity (HAT) and reduced histone deacetylase activity (HDAC), which was associated with abundant expression of acetylated histone 3 and 4 (Ac-H3 and Ac-H4, respectively). Moreover, Ac-H3 and Ac-H4 readily co-localized with the spontaneous and mitomycin C-induced DSBs in BCR-ABL1 –positive leukemia cells suggesting that histone acetylation and chromatin re-modeling is important for efficient repair of numerous DSBs.
BCR-ABL1; leukemia; histone acetylation; DNA repair
Patients with acute myeloid leukemia (AML) and a FLT3 internal tandem duplication (ITD) mutation have a poor prognosis, and FLT3 inhibitors are now under clinical investigation. PIM1, a serine/threonine kinase, is up-regulated in FLT3-ITD AML and may be involved in FLT3-mediated leukemogenesis. We employed a PIM1 inhibitor, AR00459339 (Array Biopharma Inc.), to investigate the effect of PIM1 inhibition in FLT3-mutant AML. Like FLT3 inhibitors, AR00459339 was preferentially cytotoxic to FLT3-ITD cells, as demonstrated in the MV4-11, Molm-14, and TF/ITD cell lines, as well as 12 FLT3-ITD primary samples. Unlike FLT3 inhibitors, AR00459339 did not suppress phosphorylation of FLT3, but did promote the de-phosphorylation of downstream FLT3 targets, STAT5, AKT, and BAD. Combining AR00459339 with a FLT3 inhibitor resulted in additive to mildly synergistic cytotoxic effects. AR00459339 was cytotoxic to FLT3-ITD samples from patients with secondary resistance to FLT3 inhibitors, suggesting a novel benefit to combining these agents. We conclude that PIM1 appears to be closely associated with FLT3 signaling, and that inhibition of PIM1 may hold therapeutic promise, either as monotherapy, or by overcoming resistance to FLT3 inhibitors.