Phase 2 study of ibrutinib in classic and variant hairy cell leukemia.

Hairy cell leukemia (HCL) is a rare B-cell malignancy, and there is a need for novel treatments for patients who do not benefit from purine analogs. Ibrutinib, an oral agent targeting Bruton tyrosine kinase in the B-cell receptor signaling pathway, is highly effective in several malignancies. Its activity in HCL was unknown, so we conducted a multisite phase 2 study of oral ibrutinib in patients with either relapsed classic or variant hairy cell leukemia. The primary outcome measure was the overall response rate (ORR) at 32 weeks, and we also assessed response at 48 weeks and best response during treatment. Key secondary objectives were characterization of toxicity and determination of progression-free survival (PFS) and overall survival (OS). Thirty-seven patients were enrolled at 2 different doses (24 at 420 mg, 13 at 840 mg). The median duration of follow-up was 3.5 years (range, 0-5.9 years). The ORR at 32 weeks was 24%, which increased to 36% at 48 weeks. The best ORR was 54%. The estimated 36-month PFS was 73% and OS was 85%. The most frequent adverse events were diarrhea (59%), fatigue (54%), myalgia (54%), and nausea (51%). Hematologic adverse events were common: anemia (43%), thrombocytopenia (41%), and neutropenia (35%). Ibrutinib can be safely administered to patients with HCL with objective responses and results in prolonged disease control. Although the initial primary outcome objective of the study was not met, the observation of objective responses in heavily pretreated patients coupled with a favorable PFS suggests that ibrutinib may be beneficial in these patients. This trial was registered at www.clinicaltrials.gov as #NCT01841723.

Transducin ß-like protein 1 controls multiple oncogenic networks in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common Non-Hodgkin's lymphoma and is characterized by a remarkable heterogeneity with diverse variants that can be identified histologically and molecularly. Large-scale gene expression profiling studies have identified the germinal center B-cell (GCB-) and activated B-cell (ABC-) subtypes. Standard chemo-immunotherapy remains standard front line therapy, curing approximately two thirds of patients. Patients with refractory disease or those who relapse after salvage treatment have an overall poor prognosis highlighting the need for novel therapeutic strategies. Transducin ß-like protein 1 (TBL1) is an exchange adaptor protein encoded by the TBL1X gene and known to function as a master regulator of the Wnt signalling pathway by binding to ß-CATENIN and promoting its downstream transcriptional program. Here, we show that, unlike normal B-cells, DLBCL cells express abundant levels of TBL1 and its overexpression correlates with poor clinical outcome regardless of DLBCL molecular subtype. Genetic deletion of TBL1 and pharmacological approach using tegavivint, a first-in-class small molecule targeting TBL1 (Iterion Therapeutics), promotes DLBCL cell death in vitro and in vivo. Through an integrated genomic, biochemical, and pharmacologic analyses, we characterized a novel, ß-CATENIN independent, post-transcriptional oncogenic function of TBL1 in DLBCL where TBL1 modulates the stability of key oncogenic proteins such as PLK1, MYC, and the autophagy regulatory protein BECLIN-1 through its interaction with a SKP1-CUL1-F-box (SCF) protein supercomplex. Collectively, our data provide the rationale for targeting TBL1 as a novel therapeutic strategy in DLBCL.

p53-/- synergizes with enhanced NrasG12D signaling to transform megakaryocyte-erythroid progenitors in acute myeloid leukemia.

Somatic mutations in TP53 and NRAS are associated with transformation of human chronic myeloid diseases to acute myeloid leukemia (AML). Here, we report that concurrent RAS pathway and TP53 mutations are identified in a subset of AML patients and confer an inferior overall survival. To further investigate the genetic interaction between p53 loss and endogenous NrasG12D/+ in AML, we generated conditional NrasG12D/+p53-/- mice. Consistent with the clinical data, recipient mice transplanted with NrasG12D/+p53-/- bone marrow cells rapidly develop a highly penetrant AML. We find that p53-/- cooperates with NrasG12D/+ to promote increased quiescence in megakaryocyte-erythroid progenitors (MEPs). NrasG12D/+p53-/- MEPs are transformed to self-renewing AML-initiating cells and are capable of inducing AML in serially transplanted recipients. RNA sequencing analysis revealed that transformed MEPs gain a partial hematopoietic stem cell signature and largely retain an MEP signature. Their distinct transcriptomes suggests a potential regulation by p53 loss. In addition, we show that during AML development, transformed MEPs acquire overexpression of oncogenic Nras, leading to hyperactivation of ERK1/2 signaling. Our results demonstrate that p53-/- synergizes with enhanced oncogenic Nras signaling to transform MEPs and drive AML development. This model may serve as a platform to test candidate therapeutics in this aggressive subset of AML.

Decitabine enhances anti-CD33 monoclonal antibody BI 836858-mediated natural killer ADCC against AML blasts.

Acute myeloid leukemia (AML) is the most common type of acute leukemia, affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence, novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to improve overall survival, validating CD33 as a target for antibody-based therapy of AML. Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents that show efficacy in older patients, did not compromise BI 836858-induced NK-cell-mediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples compared with pre-DAC samples; when NKG2DL receptor was blocked using antibodies, BI 836858-mediated ADCC was significantly decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML.

The interleukin-3 receptor CD123 targeted SL-401 mediates potent cytotoxic activity against CD34+CD123+ cells from acute myeloid leukemia/myelodysplastic syndrome patients and healthy donors.

Diseases with clonal hematopoiesis such as myelodysplastic syndrome and acute myeloid leukemia have high rates of relapse. Only a small subset of acute myeloid leukemia patients are cured with chemotherapy alone. Relapse in these diseases occurs at least in part due to the failure to eradicate leukemic stem cells or hematopoietic stem cells in myelodysplastic syndrome. CD123, the alpha chain of the interleukin-3 receptor heterodimer, is expressed on the majority of leukemic stem cells and myelodysplastic syndrome hematopoietic stem cells and in 80% of acute myeloid leukemia. Here, we report indiscriminate killing of CD123+ normal and acute myeloid leukemia / myelodysplastic syndrome cells by SL-401, a diphtheria toxin interleukin-3 fusion protein. SL-401 induced cytotoxicity of CD123+ primary cells/blasts from acute myeloid leukemia and myelodysplastic syndrome patients but not CD123- lymphoid cells. Importantly, SL-401 was highly active even in cells expressing low levels of CD123, with minimal effect on modulation of the CD123 target in acute myeloid leukemia. SL-401 significantly prolonged survival of leukemic mice in acute myeloid leukemia patient-derived xenograft mouse models. In addition to primary samples, studies on normal cord blood and healthy marrow show that SL-401 has activity against normal hematopoietic progenitors. These findings indicate potential use of SL-401 as a "bridge-to-transplant" before allogeneic hematopoietic cell transplantation in acute myeloid leukemia / myelodysplastic syndrome patients.

Prognostic and biologic significance of long non-coding RNA profiling in younger adults with cytogenetically normal acute myeloid leukemia.

Long non-coding ribonucleic acids (RNAs) are a novel class of RNA molecules, which are increasingly recognized as important molecular players in solid and hematologic malignancies. Herein we investigated whether long non-coding RNA expression is associated with clinical and molecular features, as well as outcome of younger adults (aged <60 years) with de novo cytogenetically normal acute myeloid leukemia. Whole transcriptome profiling was performed in a training (n=263) and a validation set (n=114). Using the training set, we identified 24 long non-coding RNAs associated with event-free survival. Linear combination of the weighted expression values of these transcripts yielded a prognostic score. In the validation set, patients with high scores had shorter disease-free (P<0.001), overall (P=0.002) and event-free survival (P<0.001) than patients with low scores. In multivariable analyses, long non-coding RNA score status was an independent prognostic marker for disease-free (P=0.01) and event-free survival (P=0.002), and showed a trend for overall survival (P=0.06). Among multiple molecular alterations tested, which are prognostic in cytogenetically normal acute myeloid leukemia, only double CEBPA mutations, NPM1 mutations and FLT3-ITD associated with distinct long non-coding RNA signatures. Correlation of the long non-coding RNA scores with messenger RNA and microRNA expression identified enrichment of genes involved in lymphocyte/leukocyte activation, inflammation and apoptosis in patients with high scores. We conclude that long non-coding RNA profiling provides meaningful prognostic information in younger adults with cytogenetically normal acute myeloid leukemia. In addition, expression of prognostic long non-coding RNAs associates with oncogenic molecular pathways in this disease. clinicaltrials.gov Identifier: 00048958 (CALGB-8461), 00899223 (CALGB-9665), and 00900224 (CALGB-20202).

Cardiac Glycoside Constituents of Streblus asper with Potential Antineoplastic Activity.

Three new (1-3) and two known (4 and 5) cytotoxic cardiac glycosides were isolated and characterized from a medicinal plant, Streblus asper Lour. (Moraceae), collected in Vietnam, with six new analogues and one known derivative (5a-g) synthesized from (+)-strebloside (5). A preliminary structure-activity relationship study indicated that the C-10 formyl and C-5 and C-14 hydroxy groups and C-3 sugar unit play important roles in the mediation of the cytotoxicity of (+)-strebloside (5) against HT-29 human colon cancer cells. When evaluated in NCr nu/nu mice implanted intraperitoneally with hollow fibers facilitated with either MDA-MB-231 human breast or OVCAR3 human ovarian cancer cells, (+)-strebloside (5) showed significant cell growth inhibitory activity in both cases, in the dose range 5-30 mg/kg.

Persistence of DNMT3A R882 mutations during remission does not adversely affect outcomes of patients with acute myeloid leukaemia.

Somatic mutation of the DNMT3A gene at the arginine R882 site is common in acute myeloid leukaemia (AML). The prognostic significance of DNMT3A R882 mutation clearance, using traditional diagnostic next generation sequencing (NGS) methods, during complete remission (CR) in AML patients is controversial. We examined the impact of clearing DNMT3A R882 mutations at diagnosis to the detectable threshold of ˂3% during CR on outcome in 56 adult AML patients. Mutational remission, defined as clearance of pre-treatment DNMT3A R882 and all other AML-associated mutations to a variant allele frequency ˂3%, occurred in 14 patients whereas persistent DNMT3A R882 mutations were observed in 42 patients. There were no significant differences in disease-free or overall survival between patients with and without DNMT3A R882 mutation clearance. Patients with persistent DNMT3A R882 who cleared all other AML mutations and did not acquire new mutations (n = 30), trended towards longer disease-free survival (1·6 vs. 0·6 years, P = 0·06) than patients with persistence of DNMT3A R882, in addition to other mutations or acquisition of new AML-associated mutations, such as those in TET2, JAK2, ASXL1 and TP53 (n = 12). These data demonstrate that DNMT3A R882 mutations, as assessed by traditional NGS methods, persist in the majority of AML patients in CR.

Daunorubicin-Loaded DNA Origami Nanostructures Circumvent Drug-Resistance Mechanisms in a Leukemia Model.

Many cancers show primary or acquired drug resistance due to the overexpression of efflux pumps. A novel mechanism to circumvent this is to integrate drugs, such as anthracycline antibiotics, with nanoparticle delivery vehicles that can bypass intrinsic tumor drug-resistance mechanisms. DNA nanoparticles serve as an efficient binding platform for intercalating drugs (e.g., anthracyclines doxorubicin and daunorubicin, which are widely used to treat acute leukemias) and enable precise structure design and chemical modifications, for example, for incorporating targeting capabilities. Here, DNA nanostructures are utilized to circumvent daunorubicin drug resistance at clinically relevant doses in a leukemia cell line model. The fabrication of a rod-like DNA origami drug carrier is reported that can be controllably loaded with daunorubicin. It is further directly verified that nanostructure-mediated daunorubicin delivery leads to increased drug entry and retention in cells relative to free daunorubicin at equal concentrations, which yields significantly enhanced drug efficacy. Our results indicate that DNA origami nanostructures can circumvent efflux-pump-mediated drug resistance in leukemia cells at clinically relevant drug concentrations and provide a robust DNA nanostructure design that could be implemented in a wide range of cellular applications due to its remarkably fast self-assembly (≈5 min) and excellent stability in cell culture conditions.

Validation of ZAP-70 methylation and its relative significance in predicting outcome in chronic lymphocytic leukemia.

ZAP-70 methylation 223 nucleotides downstream of transcription start (CpG+223) predicts outcome in chronic lymphocytic leukemia (CLL), but its impact relative to CD38 and ZAP-70 expression or immunoglobulin heavy chain variable region (IGHV) status is uncertain. Additionally, standardizing ZAP-70 expression analysis has been unsuccessful. CpG+223 methylation was quantitatively determined in 295 untreated CLL cases using MassARRAY. Impact on clinical outcome vs CD38 and ZAP-70 expression and IGHV status was evaluated. Cases with low methylation (<20%) had significantly shortened time to first treatment (TT) and overall survival (OS) (P < .0001). For TT, low methylation defined a large subset of ZAP-70 protein-negative cases with significantly shortened TT (median, 8.0 vs 3.9 years for high vs low methylation; hazard ratio [HR] = 0.43; 95% confidence interval [CI], 0.25-0.74). Conversely, 16 ZAP-70 protein-positive cases with high methylation had poor outcome (median, 1.1 vs 2.3 years for high vs low methylation; HR = 1.62; 95% CI, 0.87-3.03). For OS, ZAP-70 methylation was the strongest risk factor; CD38 and ZAP-70 expression or IGHV status did not significantly improve OS prediction. A pyrosequencing assay was established that reproduced the MassARRAY data (κ coefficient > 0.90). Thus, ZAP-70 CpG+223 methylation represents a superior biomarker for TT and OS that can be feasibly measured, supporting its use in risk-stratifying CLL.

Proteomic profiling identifies specific histone species associated with leukemic and cancer cells.

BACKGROUND: Chromatin is an extraordinarily complex structure. Much of this complexity results from the presence of numerous histone post-translational modifications and histone variants. Alterations in the patterns of histone post-translational modifications are emerging as a feature of many types of cancer and have been shown to have prognostic value. RESULTS: We have applied a liquid chromatography/mass spectrometry-based approach to comprehensively characterize the histone proteome in primary samples from chronic lymphocytic leukemia (CLL) patients, as well as bladder and breast cancer cell culture models. When compared to non-malignant CD19+ B cells from healthy donors, the CLL histone proteome showed a distinct signature of differentially expressed species, spanning all the histones studied and including both post-translationally modified species and unmodified, non-allelic replication-dependent histone isoforms. However, the large changes in histone H3 and H4 that are characteristic of many cancer types were not observed. One of species of H2A (mass = 14,063 Da) was the most strongly associated with time to treatment in CLL patients. CLL patient samples also demonstrated histone profiles that were distinct from those of the bladder and breast cancer cells. CONCLUSIONS: Signatures of histone profiles are complex and can distinguish between healthy individuals and CLL patients and may provide prognostic markers. In addition, histone profiles may define tissue specific malignancies.

Cotreatment of hairy cell leukemia and melanoma with the BRAF inhibitor dabrafenib.

The activating BRAF mutation p.V600E has been identified in many cancers, including colon and lung adenocarcinomas, papillary thyroid cancer, malignant melanoma, and hairy cell leukemia (HCL). Malignant melanoma and HCL are of particular interest because of both the high proportion of cases harboring the mutation and the dramatic responses to BRAF inhibitor therapy reported in the literature. This report presents a patient with HCL and malignant melanoma with the BRAF p.V600E mutation, and discusses the successful treatment of both cancers with the BRAF inhibitor dabrafenib.

Increasing the complexity of chromatin: functionally distinct roles for replication-dependent histone H2A isoforms in cell proliferation and carcinogenesis.

Replication-dependent histones are encoded by multigene families found in several large clusters in the human genome and are thought to be functionally redundant. However, the abundance of specific replication-dependent isoforms of histone H2A is altered in patients with chronic lymphocytic leukemia. Similar changes in the abundance of H2A isoforms are also associated with the proliferation and tumorigenicity of bladder cancer cells. To determine whether these H2A isoforms can perform distinct functions, expression of several H2A isoforms was reduced by siRNA knockdown. Reduced expression of the HIST1H2AC locus leads to increased rates of cell proliferation and tumorigenicity. We also observe that regulation of replication-dependent histone H2A expression can occur on a gene-specific level. Specific replication-dependent histone H2A genes are either up- or downregulated in chronic lymphocytic leukemia tumor tissue samples. In addition, discreet elements are identified in the 5' untranslated region of the HIST1H2AC locus that confer translational repression. Taken together, these results indicate that replication-dependent histone isoforms can possess distinct cellular functions and that regulation of these isoforms may play a role in carcinogenesis.

Rocaglamide, silvestrol and structurally related bioactive compounds from Aglaia species.

Covering: 2006 to 2013. Investigations on the chemistry and biology of rocaglamide, silvestrol and structurally related bioactive compounds from Aglaia species during the period 2006-2013 are reviewed. Included are new phytochemical studies of naturally occurring rocaglamide derivatives, an update on synthetic methods for cyclopenta[b]benzofurans, and a description of the recent biological evaluation and mechanism-of-action studies on compounds of this type.

ER stress and autophagy: new discoveries in the mechanism of action and drug resistance of the cyclin-dependent kinase inhibitor flavopiridol.

Cyclin dependent kinase (CDK) inhibitors, such as flavopiridol, demonstrate significant single-agent activity in chronic lymphocytic leukemia (CLL), but the mechanism of action in these nonproliferating cells is unclear. Here we demonstrate that CLL cells undergo autophagy after treatment with therapeutic agents, including fludarabine, CAL-101, and flavopiridol as well as the endoplasmic reticulum (ER) stress-inducing agent thapsigargin. The addition of chloroquine or siRNA against autophagy components enhanced the cytotoxic effects of flavopiridol and thapsigargin, but not the other agents. Similar to thapsigargin, flavopiridol robustly induces a distinct pattern of ER stress in CLL cells that contributes to cell death through IRE1-mediated activation of ASK1 and possibly downstream caspases. Both autophagy and ER stress were documented in tumor cells from CLL patients receiving flavopiridol. Thus, CLL cells undergo autophagy after multiple stimuli, including therapeutic agents, but only with ER stress mediators and CDK inhibitors is autophagy a mechanism of resistance to cell death. These findings collectively demonstrate, for the first time, a novel mechanism of action (ER stress) and drug resistance (autophagy) for CDK inhibitors, such as flavopiridol in CLL, and provide avenues for new therapeutic combination approaches in this disease.

Silencing of the inhibitor of DNA binding protein 4 (ID4) contributes to the pathogenesis of mouse and human CLL.

Inhibitor of DNA binding protein 4 (ID4) is a member of the dominant-negative basic helix-loop-helix transcription factor family that lacks DNA binding activity and has tumor suppressor function. ID4 promoter methylation has been reported in acute myeloid leukemia and chronic lymphocytic leukemia (CLL), although the expression, function, and clinical relevance of this gene have not been characterized in either disease. We demonstrate that the promoter of ID4 is consistently methylated to various degrees in CLL cells, and increased promoter methylation in a univariable analysis correlates with shortened patient survival. However, ID4 mRNA and protein expression is uniformly silenced in CLL cells irrespective of the degree of promoter methylation. The crossing of ID4(+/-) mice with Eµ-TCL1 mice triggers a more aggressive murine CLL as measured by lymphocyte count and inferior survival. Hemizygous loss of ID4 in nontransformed TCL1-positive B cells enhances cell proliferation triggered by CpG oligonucleotides and decreases sensitivity to dexamethasone-mediated apoptosis. Collectively, this study confirms the importance of the silencing of ID4 in murine and human CLL pathogenesis.

AP-1 elements and TCL1 protein regulate expression of the gene encoding protein tyrosine phosphatase PTPROt in leukemia.

We previously demonstrated that the gene encoding PTPROt, the truncated form of protein tyrosine phosphatase receptor type O expressed predominantly in hematopoietic cells, is a candidate tumor suppressor and is down-regulated in chronic lymphocytic leukemia (CLL). Here, we show that PTPROt expression is significantly reduced in CD19(+) spleen B cells from Eµ-T cell leukemia 1 (TCL1) transgenic mice relative to the wild-type mice. Strikingly, as much as a 60% decrease in PTPROt expression occurs at 7 weeks independently of promoter methylation. To elucidate the potential mechanism for this early suppression of PTPROt in these mice, we explored the role of activating protein-1 (AP-1) in its expression. We first demonstrate that AP-1 activation by 12-O-tetradecanoylphorbol-13-acetate induces PTPROt expression with concurrent recruitment of c-fos and c-jun to its promoter. The PTPROt promoter is also responsive to over- and underexpression of AP-1, confirming the role of AP-1 in PTPROt expression. Next, we demonstrate that TCL1 can repress the PTPROt promoter by altering c-fos expression and c-jun activation state. Finally, using primary CLL cells we have shown an inverse relationship between TCL1 and PTPROt expression. These findings further substantiate the role of TCL1 in PTPROt suppression and its importance in the pathogenesis of CLL.

Bioactive constituents of Indigofera spicata.

Four new flavanones, designated as (+)-5″-deacetylpurpurin (1), (+)-5-methoxypurpurin (2), (2S)-2,3-dihydrotephroglabrin (3), and (2S)-2,3-dihydrotephroapollin C (4), together with two known flavanones (5 and 6), three known rotenoids (7-9), and one known chalcone (10) were isolated from a chloroform-soluble partition of a methanol extract from the combined flowers, fruits, leaves, and twigs of Indigofera spicata, collected in Vietnam. The compounds were obtained by bioactivity-guided isolation using the HT-29 human colon cancer, 697 human acute lymphoblastic leukemia, and Raji human Burkitt's lymphoma cell lines. The structures of 1-4 were established by extensive 1D- and 2D-NMR experiments, and the absolute configurations were determined by the measurement of specific rotations and CD spectra. The cytotoxic activities of the isolated compounds were tested against the HT-29, 697, Raji, and CCD-112CoN human normal colon cells. Also, the quinone reductase induction activities of the isolates were determined using the Hepa 1c1c7 murine hepatoma cell line. In addition, cis-(6aß,12aß)-hydroxyrotenone (7) was evaluated in an in vivo hollow fiber bioassay using HT-29, MCF-7 human breast cancer, and MDA-MB-435 human melanoma cells.