Acceleration of AML Progression By Cigarette Smoke Exposure or Condensate Exposure and Associated DNA Methylation Alterations

Subsets of acute myelogenous leukemia (AML) are characterized by molecular alterations with prognostic significance, however, little is known about how modifiable behaviors, such as cigarette smoking, intersect with genetic factors. Mutations rendering the Fms-like tyrosine kinase 3 (FLT3) constitutively active, such as internal tandem duplication (ITD), are associated with refractory disease and therapy resistance. Inhibition of the FLT3 kinase shows some benefit in this population, as highlighted by the FDA approvals of midostaurin and gilteritinib, but overall outcomes remain poor. Cigarette smoke exposure (CSE) also marks a population of patients with poor prognosis. Current and former smokers who develop AML are known to have worse survival as compared to never smokers (Alfayez M., et al. ASCO 2019), but the impact of FLT3 mutation and subsequent associated treatment response has not been studied. Also, the underlying mechanism of how history of cigarette smoking influences leukemia biology and response to therapy is poorly understood. In order to model a history of smoking in AML patients, NOD-SCID mice (n=25) were exposed to CSE using a smoking robot for 2 hours, 5 days/week, for 2 weeks. Mice were then inoculated via tail-vein injection with luciferase tagged human FLT3-ITD cells and leukemia burden was monitored through noninvasive imaging. CSE continued through the duration of the experiment, post engraftment. Control "non-smoking" mice (n=15) were only injected with leukemia cells. Within one week post leukemic introduction, a significant increase in leukemic burden as measured by bioluminescence was apparent in mice exposed to CSE versus control mice (P-value<0.0001). To model the impact of smoking cessation upon diagnosis of AML in patients, experiments were modified to halt CSE once leukemic engraftment was detectable by non-invasive imaging. Smoking cessation versus continuous smoke exposure yielded reduced relative leukemic burden. Mice with continuous smoke exposure had higher rates of leukemia compared to mice who ceased smoking (n=10) one week prior (P-value =0.0064). These rapid changes in leukemic burden suggest that CSE may prime the microenvironment to promote leukemia progression or directly affect leukemia cells. To address the latter possibility, human AML cells were exposed to cigarette smoke condensate (CSC), which contains the chemicals present in cigarettes, for two weeks before introducing the cells into mice. A significant increase in leukemic burden was observed in mice injected cells exposed to CSC compared to mice injected with unexposed leukemia cells (P-value <0.001), highlighting a direct role for the chemicals in cigarettes on in vivo leukemia proliferative factors. Smokers are known to carry altered global DNA methylation signatures that persist decades after quitting. To measure DNA methylation changes in the in vivo models described above, we examined spleens of non-smoking and smoke exposed mice by reduced representative bisulfite sequencing (RRBS). Sequences were mapped to either the human or mouse genome, (enabling identification of leukemia specific versus microenvironment specific alterations) and were compared in the smoking and non-smoking mice. Over 200 genes exhibited significant DNA methylation alterations in their promoter regions. Genes involved in RNA polymerase activity and chromatin remodeling were highly represented amongst those with altered DNA methylation. The clinical significance of our observations was confirmed in a cohort of 58 treatment naïve FLT3-ITD AML patients at MD Anderson receiving intensive induction therapy: 41 never smokers and 17 ever smokers. Smokers had significantly reduced survival as compared to the never smokers (median overall survival of 18 vs 23 months, P-value 0.0092). Collectively our findings indicate that short-term CSE is sufficient to alter DNA methylation patterns and accelerate the early progression of FLT3-ITD AML in vivo. Smoking cessation upon diagnosis may slow leukemic growth relative to smoking throughout AML therapy prompting the consideration of behavioral interventions for smokers with AML. Improved understanding of the mechanism of leukemic progression and drug resistance from CSE is expected to lead to improved treatment paradigms designed for patients with a history of cigarette smoking. Disclosures Konopleva: Genentech: Honoraria, Research Funding; Kisoji: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Ascentage: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding. Jabbour:Cyclacel LTD: Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. DiNardo:agios: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; jazz: Honoraria; medimmune: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; daiichi sankyo: Honoraria; syros: Honoraria; celgene: Consultancy, Honoraria.

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Cigarette Smoke or Cigarette Condensate Exposure Accelerates Growth of FLT3-ITD AML Models, Induces Oxidative Stress, and Alters DNA Methylation

Blood ◽

10.1182/blood-2021-145944 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3331-3331

Author(s):

Mary Figueroa ◽

Megan R Rodriguez ◽

Marcos Estecio ◽

Yue Lu ◽

Seyed Javad Moghaddam ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Methylation ◽

Cigarette Smoking ◽

Cigarette Smoke ◽

Intellectual Property Rights ◽

Research Funding ◽

P Value ◽

Grant Support ◽

History Of ◽

Support Research

Abstract Acute myeloid leukemia (AML) patients with any history of cigarette smoking have worse survival outcomes compared to patients that have never smoked. The molecular basis of cigarette smoking or cigarette smoke exposure (CSE) impacting AML progression or treatment response is unknown. Altered DNA methylation from smoking persists decades after quitting and has been followed in peripheral blood mononuclear cells (PBMCs) but have not been evaluated in the context of AML. Smoking also causes oxidative stress in PBMCs, but this has yet to be studied in AML patients with a history of smoking. To model how smoking worsens AML progression, we created a CSE model using a cigarette smoking robot where NOD-SCID mice received 2 hours of CSE 5 days/week or air alone. After 2 weeks of CSE, 100,000 luciferase-tagged, human AML cells were injected via tail-vein and leukemic burden was monitored by bioluminescent imaging. Two cell lines, MOLM13 and MOLM14 carrying the oncogenic fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation, when introduced into CSE mice had enhanced leukemic progression within one week (p-value <0.0001 and <0.001 respectively). MOLM14-bearing mice showed increased leukemic burden 24 days post injection (p-value<0.05). DNA methylation was evaluated by bisulfite sequencing of splenocytes from CSE mice, which was mapped to the human (AML) or mouse (host) genome. Over 200 significant changes in DNA methylation of gene promoters was seen, including hypomethylation of aryl-hydrocarbon receptor repressor (AHRR), an established hallmark of smoking in humans. Indicating that our CSE model recapitulates DNA methylation from smoking in humans. Additionally, GATA-2, a critical protein for hematopoietic differentiation known to be frequently mutated in AML, was also amongst the top hypomethylated genes. We quarried TCGA to understand the implications of altered DNA methylation in AML patients. In AML patients, low GATA-2 methylation showed decreased survival compared to those with high GATA-2 methylation (N=42/group, p-value: 0.000138). The discovery of GATA-2 methylation in smoking models and its attribution as a poor prognostic indicator in AML is novel, which underscores a need to understand the role of GATA-2 methylation in AML. Reactive oxygen species (ROS) have been implicated in AML progression, drug resistance, and are elevated in otherwise healthy smokers. No significant differences were seen in intracellular ROS in spleen or PBMCs of CSE mice; however, we found more than a two-fold increase of heme oxygenase 1 (HO-1) (p-value:0.0505), a protein involved in antioxidant responses and AML treatment resistance. There was also increased BCL-2 protein expression and a decrease in AHRR expression (p-value: 0.0098) in CSE mouse samples. This suggests that CSE causes oxidative stress and increases pro-leukemic signaling. To address if CSE impacted treatment efficacy, we treated mice with daunorubicin (2 mg/kg, twice weekly via tail-vein) once evidence of engraftment was detected. We found a trend towards increased leukemic burden compared to non-smoking mice which approached statistical significance. To study the direct impact of CSE on AML, without exposure to the tumor environment, AML cells were treated in vitro with a commercially available cigarette smoke condensate (CSC) that contains the chemicals from cigarette smoke. To mimic the in vivo CSE, MOLM13 cells received two weeks of CSC treatment before being injected into mice. At 3, 10, and 17 days post injection, mice with CSC-treated AML had enhanced leukemic burden (p-value <0.0001, <0.0001, and <0.001). This model revealed that chemicals in cigarette smoke can directly promote AML. On day 14 of CSC treatment, mirroring when the cells were injected into mice, both FLT3-ITD cell lines had increased ROS levels and or glutathione as measured by flow cytometry; this is indicative of CSC-induced oxidative stress. Cumulatively, these data define novel changes in DNA methylation and redox from smoking or tobacco products with strong potential to drive AML progression and therapy resistance. Future studies will determine if blocking these redox or methylation events can reverse the accelerated AML growth and will aid in the creation of a tailored treatment strategy for AML patients with any history of smoking. Disclosures Jabbour: Amgen, AbbVie, Spectrum, BMS, Takeda, Pfizer, Adaptive, Genentech: Research Funding. Konopleva: Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; AstraZeneca: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; Sanofi: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; KisoJi: Research Funding; Stemline Therapeutics: Research Funding; Agios: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Cellectis: Other: grant support; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding. DiNardo: Novartis: Honoraria; Agios/Servier: Consultancy, Honoraria, Research Funding; Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; ImmuneOnc: Honoraria, Research Funding; Takeda: Honoraria; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding.

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Cigarette Smoke or Cigarette Condensate Exposure Enhances Growth of FLT3-ITD AML Models and Alters DNA Methylation and Leukemic Gene Expression

Blood ◽

10.1182/blood-2020-142478 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 29-30

Author(s):

Mary Figueroa ◽

Maninder Khosla ◽

Yue Lu ◽

Marcos Estecio ◽

Seyed Javad Moghaddam ◽

...

Keyword(s):

Dna Methylation ◽

Cigarette Smoke ◽

Research Funding ◽

Smoke Exposure ◽

Scid Mice ◽

Cigarette Smoke Exposure ◽

P Value ◽

The Impact ◽

Advisory Role

Current and former smoker AML patients have worse survival outcomes compared to never smokers. This worsened prognosis of smokers with AML can also be seen in patients who carry activating mutations of the Fms-like tyrosine kinase 3 (FLT3) and are treated with regimens that include newly approved kinase inhibitors. While the impact of genetic mutations on survival in AML have been studied, and some have been therapeutically targeted, the role of cigarette smoking or cigarette smoke exposure (which is potentially modifiable) on leukemia progression or treatment response is understudied. In order to elucidate molecular effects of cigarette smoke exposure (CSE) that contribute to the poor prognosis of AML patients, we developed a cigarette smoke exposure model for mice to mimic the current and former smoking habits of AML patients. NOD-SCID mice were exposed to CSE in a smoking robot for 2 hours, 5 days/week, for 2 weeks or to air alone as a control. Mice were then injected with luciferase-tagged human AML cell lines, and leukemic burden was monitored through non-invasive bioluminescent imaging. Control "non-smoking" mice were only subject to AML cell injection. Enhanced early leukemic-burden was observed two distinct FLT3-ITD AML models, MOLM13 and MOLM14, within one week post AML introduction (p-value <0.0001 and <0.001 respectively). Although the latter model showed slightly longer latency of disease with increased leukemic burden apparent 24 days post leukemic introduction (p-value <0.05). In order to address if the early increase in leukemic burden may have arisen from extrinsic factors in the tumor microenvironment, we utilized non-leukemia bearing immunocompetent mice exposed to CSE using the 2 week exposure scheme and saw enhanced myeloid progenitor growth, indicating evidence of microenvironment priming of myeloid cells by CSE. One month of CSE increased the MPP1 and MPP2 populations in the bone marrow of NOD-SCID mice. C57BL/6J mice had increased myeloid and hematopoietic stem cell populations after a month of CSE (p-value <0.05). We also modeled the effect of smoking cessation upon leukemia engraftment by halting smoke exposure compared to mice that continued smoking. Cessation significantly slowed leukemic growth in MOLM13 bearing mice (N=10, p-value<0.01). Cigarette smoke exposure globally alters DNA methylation in blood cells and these changes can persist for decades. Independent of mutations, DNA methylation patterns in AML patients have prognostic significance. To understand how CSE accelerated leukemic growth in vivo, DNA methylation was evaluated using reduced representative bisulfite sequencing. More than two hundred significant alterations in DNA methylation across the promoter region of genes were found AML cells from spleen samples of CSE MOLM13-bearing mice as compared to non-smoking mice. Among the genes with the most significantly altered DNA methylation were GATA-2, an important protein for hematopoietic differentiation, and aryl-hydrocarbon receptor repressor (AHRR), a gene whose hypomethylation is a hallmark of cigarette smoke exposure. To identify the impact of cigarette smoke exposure on the leukemia cells in the absence of the tumor microenvironment we treated AML cells directly using a cigarette smoke condensate (CSC) that contains the chemicals in cigarette smoke used in the previously described CSE model. MOLM13 cells either treated with DMSO or 10ug/ml CSC every passage for two weeks were injected into NOD-SCID mice. This model resulted in enhanced leukemic burden 3, 10, and 17 days after leukemic introduction (p-value <0.0001, <0.0001, and <0.001) indicating strong pro-leukemic effects of CSC. Evaluation of in vitro CSC treated AML cells was conducted to identify causes for the enhanced leukemic burden. While CSC treatment yielded no changes in proliferation or survival of the cells over the course of two months, within one week there was increased expression of DNMT1 in several cells lines. Increased basal and maximal oxygen consumption, and modulation of the antioxidant gene, HO-1, was also observed along with modulation of AHRR and GATA-2, reinforcing roles for methylation data gained from in vivo CSE experiments. Discovering the mechanisms promoting AML progression from cigarette smoke exposure will lead to improved, tailored treatment for AML patients with smoking histories and our further studies of these gene changes will aid in that endeavor. Disclosures Jabbour: Takeda: Other: Advisory role, Research Funding; AbbVie: Other: Advisory role, Research Funding; Amgen: Other: Advisory role, Research Funding; Pfizer: Other: Advisory role, Research Funding; Genentech: Other: Advisory role, Research Funding; BMS: Other: Advisory role, Research Funding; Adaptive Biotechnologies: Other: Advisory role, Research Funding. Konopleva:Genentech: Consultancy, Research Funding; Ascentage: Research Funding; Forty-Seven: Consultancy, Research Funding; Calithera: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Ablynx: Research Funding; Agios: Research Funding; Amgen: Consultancy; AstraZeneca: Research Funding; Eli Lilly: Research Funding; Kisoji: Consultancy; Cellectis: Research Funding; Rafael Pharmaceutical: Research Funding; AbbVie: Consultancy, Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Sanofi: Research Funding. DiNardo:Agios: Consultancy, Honoraria, Research Funding; Syros: Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Calithera: Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Notable Labs: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; MedImmune: Honoraria; ImmuneOnc: Honoraria; Jazz: Honoraria.

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Integrated Analysis of Global DNA Methylation and Transcription Reveals a Leukemia Subtype with Extreme Hypermethylation Associated with Silencing of Regulators of Differentiation

Blood ◽

10.1182/blood-2018-99-118521 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2608-2608

Author(s):

Claudia Gebhard ◽

Roger Mulet-Lazaro ◽

Lucia Schwarzfischer ◽

Dagmar Glatz ◽

Margit Nuetzel ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Promoter Methylation ◽

Research Funding ◽

Promoter Hypermethylation ◽

Ctcf Binding ◽

P Value ◽

Global Dna Methylation ◽

Employment Equity ◽

Equity Ownership

Abstract Acute myeloid leukemia (AML) represents a highly heterogeneous myeloid stem cell disorder classified based on various genetic defects. Besides genetic alterations, epigenetic changes are recognized as an additional mechanism contributing to leukemogenesis, but insight into the latter process remains minor. Using a combination of Methyl-CpG-Immunoprecipitation (MCIp-chip) and MALDI-TOF analysis of bisulfite-treated DNA in a cohort of 196 AML patients we previously demonstrated that (cyto)genetically defined AML subtypes, including CBFB-MYH11, AML-ETO, NPM1-mut, CEBPA-mut or IDH1/2-mut subtypes, express specific DNA-methylation profiles (Gebhard et al, Leukemia, 2018). A fraction of AML patients (5/196) displayed a unique abnormal hypermethylation profile that was completely distinct from any other AML subtype. These patients present immature leukemia (FAB M0, M1) with various chromosomal aberrations but very few mutations (e.g. no IDH1/2, KRAS, DNMT3A) that might explain the CpG island methylator phenotype (CIMP) phenotype. The CIMP patients showed high resemblance with a recently reported CEBPA methylated subgroup (Wouters et al, 2007 and Figueroa et al, 2009), which we confirmed by MCIp-chip and MALDI-TOF analysis. To explore the whole range of epigenetic alterations in the CIMP-AML patients we performed in-depth global DNA methylation and gene expression analyses (MCIp-seq and RNA-seq) in 45 AML and 12 CIMP patients from both studies. Principle component analysis and t-distributed stochastic neighbor embedding (t-SNE) revealed that CIMP patients express a unique DNA-methylation and gene-expression signature that separated them from all other AMLs. We could discriminate promoter methylation from non-promoter methylation by selecting MCIp-seq peaks within 3kb around TSS. Promoter hypermethylation was highly associated with repression of genes (PCC = -0.053, p-value = 0.00075). Hypermethylation of non-promoter regions was more strongly associated with upregulation of genes (PCC = 0.046, p-value = 4.613e-06). Interestingly, differentially methylated regions also showed a positive association with myeloid lineage CTCF binding sites (27% vs 18% expected, p-value < 2.2e-16 in a chi-square test of independence). Methylation of CTCF sites causes loss of CTCF binding, which has been reported to disrupt boundaries between so-called topologically associated domains (TADs), allowing enhancers located in a particular TAD to become accessible to genes in adjacent TADs and affect their transcription. Whether this is the case is under investigation. In this study we particularly focused on the role of hypermethylation of promoters in CIMP-AMLs. Promoters of many transcriptional regulators that are involved in the differentiation of myeloid lineages of which several are frequently mutated in AML were hypermethylated and repressed, including CEBPA, CEBPD, IRF8, GATA2, KLF4, MITF or MAFB. Notably, HMGA2, a critical regulator of myeloid progenitor expansion, exhibited the largest degree of CIMP promoter hypermethylation compared to the other AMLs, accompanied by a reduction in gene expression. Moreover, multiple members of the HOXB family and KLF1 (erythroid differentiation) were methylated and repressed as well. In addition, these patients frequently showed hypermethylation of many chromatin factors (e.g. LMNA, CHD7 or TET2). Hypermethylation of the TET2 promoter could result in a loss of maintenance DNA demethylation and therefore successive hypermethylation at CpG islands. We carried out regulome-capture-bisulfite sequencing on CIMP-AMLs compared to other AML samples and normal blood cell controls and confirmed methylation of the same transcription and chromatin factor promoters. We conclude that these leukemias represent very primitive HSCPs which are blocked in differentiation into multiple hematopoietic lineages, due to the absence of regulators of these lineages. Although the underlying cause for the extreme hypermethylation signature is still subject to ongoing studies, the consequence of promoter hypermethylation is silencing of key lineage regulators causing the differentiation arrest in these cells. We argue that these patients may particularly benefit from therapies that revert DNA methylation. Disclosures Ehninger: Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

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The Association Between Smoking Cessation Period and Metabolic Syndrome in Korean Men

Asia Pacific Journal of Public Health ◽

10.1177/1010539518786517 ◽

2018 ◽

Vol 30 (5) ◽

pp. 415-424 ◽

Cited By ~ 2

Author(s):

Hwang Sik Shin ◽

Jung Eun Oh ◽

Yong Jin Cho

Keyword(s):

Physical Activity ◽

Metabolic Syndrome ◽

Smoking Cessation ◽

Odds Ratio ◽

Health And Nutrition ◽

Quit Smoking ◽

Education Levels ◽

History Of ◽

Former Smokers ◽

Never Smokers

The association between smoking cessation period and metabolic syndrome (MS) is currently unknown. We studied 6032 men aged >19 years who participated in the Korean National Health and Nutrition Examination Surveys between 2010 and 2012. The risk of MS according to the amount of smoking and duration of smoking cessation was examined, and adjusted for age, amount of alcohol consumed, physical activity, body mass index, income, and education levels. Compared with never-smokers, there was a significant increase in the risk of MS among current smokers >10 pack-years and former smokers with a history of pack-years >30. The odds ratio for MS increased with smoking amount in both current and former smokers. But the risk of MS in former smokers was no longer significant after 20 years of smoking cessation adjusted for past smoking amount. Thus, to prevent MS, current smokers should quit smoking early and former smokers should continue quitting.

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Changes in Biomarkers of Exposure on Switching From a Conventional Cigarette to the glo Tobacco Heating Product: A Randomized, Controlled Ambulatory Study

Nicotine & Tobacco Research ◽

10.1093/ntr/ntaa135 ◽

2020 ◽

Author(s):

Nathan Gale ◽

Michael McEwan ◽

Oscar M Camacho ◽

George Hardie ◽

James Murphy ◽

...

Keyword(s):

Smoking Cessation ◽

Cigarette Smoking ◽

Tobacco Product ◽

Ambulatory Setting ◽

Controlled Study ◽

Control Group ◽

Clinical Trial Registration ◽

Randomized Controlled ◽

Biomarkers Of Exposure ◽

Never Smokers

Abstract Introduction Tobacco heating products (THPs) generate lower machine yields of toxicants compared to those found in conventional cigarette smoke. During use, these products are likely to expose users to lower levels of particulate matter and harmful and potentially harmful compounds compared with smoking cigarettes. Aims and Methods This randomized, controlled study is investigating whether biomarkers of exposure (BoE) to smoke toxicants are reduced when smokers switch from smoking cigarettes to using the glo THP in a naturalistic, ambulatory setting. Control groups include smokers who are abstaining from cigarette smoking and never-smokers. At a baseline study visit, 24-hour urine samples and spot blood samples were taken for BoE analysis, and exhaled carbon monoxide was also measured. N-(2-cyanoethyl) valine (CEVal) was used as a marker of compliance in subjects asked to refrain from combustible cigarette smoking. Subjects are being followed up at periodic intervals for 360 days; this article presents data following a planned interim analysis at day 90. Results In continuing smokers, BoE remained stable between baseline (day 1) and day 90. In both per-protocol and CEVal-compliant analysis populations, reductions in BoE were observed in subjects switching to using glo or undergoing smoking cessation. These reductions were statistically significant for a number of BoE when switching to glo was compared with continued smoking. Furthermore, in both populations, reductions observed in subjects switching to using glo were comparable to those seen with smoking cessation and were also to levels similar to those seen in never-smokers. Conclusion glo is a reduced-exposure tobacco product. Implications This clinical study builds on a previous 5-day confinement study and demonstrates that when smokers switched from smoking combustible cigarettes to using the glo THP in a naturalistic, ambulatory setting, their exposure to tobacco smoke toxicants was significantly decreased. For most BoE examined, this was to the same extent as that seen when a control group of smokers ceased cigarette smoking, or even to levels seen in never-smoker controls. This indicates that glo is a reduced-exposure product with the potential to be a reduced-risk tobacco product, when used by smokers whose cigarette consumption is displaced completely. Clinical trial registration ISRCTN81075760.

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Update of a Phase I/II Trial of 5-Azacytidine Prior to Gemtuzumab Ozogamicin (GO) for Patients with Relapsed Acute Myeloid Leukemia with Correlative Biomarker Studies

Blood ◽

10.1182/blood.v116.21.3286.3286 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3286-3286 ◽

Cited By ~ 1

Author(s):

Edward D. Ball ◽

Bruno C. Medeiros ◽

Larissa Balaian ◽

Tracy Roque ◽

Sue Corringham ◽

...

Keyword(s):

Phase I ◽

Myeloid Leukemia ◽

Research Funding ◽

Overall Response Rate ◽

Average Length ◽

Gemtuzumab Ozogamicin ◽

Leukemia Cells ◽

Grade 3 ◽

Age Range

Abstract Abstract 3286 Acute myeloid leukemia (AML) cells express the cell surface antigen CD33 that is a down-regulator of cell growth when ligated by a monoclonal antibody in a Syk-dependent manner. The response of AML cells to gemtuzumab ozogamicin (GO) also depends on Syk and SHP-1 expression (Leukemia 20:2093, 2006). The hypomethylating agent 5-azacytidine (5-aza) induced re-expression of Syk in some cases, therefore increasing the sensitivity of originally Syk-negative, non-responsive cells to CD33 ligation to levels of Syk-positive cells. We initiated a phase 1/2 clinical trial examining if treatment with 5-aza prior to GO is safe, efficacious, and whether in vivo responses to GO correlated with Syk expression and induction by 5-aza. Here we update the interim results of this trial (NCI registration number NCT00766116). In Phase I, 14 patients (9 males, 5 females), age range: 39–82 years [median: 66]) were treated with 75mg/m2 5-aza daily and GO in a dose-escalation manner, 4 cohorts total. The first cohort (n=3) received 5-aza for 2 days followed by GO at 3 mg/m2 on days 3 and 17; the second cohort (n=3) received 5-aza for 2 days followed by GO at 6 mg/m2 on days 3 and 17; the third cohort (n=4) received 5-aza for 4 days followed by GO at 6 mg/m2 on days 5 and 19; and the fourth cohort (n=4) at 5-aza for 6 days followed by GO at 6 mg/m2 on days 7 and 21. There were no responses in the first 2 cohorts. One patient in cohort 3 achieved CR, and 2 in cohort 4 achieved CR and CRp. Adverse events (≥ Grade 3) included febrile neutropenia 36%, infection 14%, pancytopenia 7%, dyspnea 7%, and retinopathy 7%. Average length on study (n=14) was 45 days with a mortality rate of 14% (unrelated to treatment). No dose-limiting toxicities were encountered in phase I, therefore the MTD is the dose in cohort 4. The overall response rate in evaluable patients in phase I (n=11) is 27%. Average time to ANC recovery (n=6): 30 days (range 15–42, median 33 days). In Phase II, 10 patients (5 males, 5 females), age range: 29–64 years (median 60) were treated at the MTD: 5-aza for 6 days and GO at 6 mg/m2 on days 7 and 21. 8 patients were in 1st relapse, 1 in 2nd and 1 in 3rd. There were 3 responders (2 CR, 1 CRp) in this phase, all in 1st relapse at baseline. Adverse events (≥ Grade 3) include febrile neutropenia 50%, infection 20%, increased LFTs 10%, thrombocytopenia 10%, dyspnea 10%, wheezing 10%, mucositis 10%, cough 10%, and hypoalbuminemia 10%. The average length on study (n=10) was 40 days with a mortality rate of 10% (not related to study treatment). Average time to ANC recovery in phase II (n=2): 15 days (range 12–17, median 15) with an overall response rate in evaluable patients (n=7) of 43%. The ORR for phase I/II (n=18) is 33%. 21 of the 24 patient sample pairs have been analyzed for Syk and SHP-1 expression (one patient did not have a baseline sample). Prior to therapy, Syk was expressed in 16 of 20 cases. After 5-aza treatment, Syk was re-expressed in all 4 negative cases, and increased over baseline in one case that was previously Syk +. SHP-1 was positive in 17 of the 20 cases and was re-expressed in all 3 negative cases. Leukemia cells from patients who achieved CR were Syk+ in 3 of 5 cases (the 6th hasn't been analyzed). Syk was re-expressed in the two negative cases after 5-aza. SHP-1 was expressed in 4 of 5 cases at baseline, and re-expressed in the one negative case after 5-aza. In vitro we analyzed inhibition of proliferation (for patients 1–6) or colony formation (for patients 7–24) induced by 5-aza and GO. 5-aza alone allowed 62.3+/−3.5 survival of leukemia cells and GO alone allowed survival of 59.5+/−1.7 leukemia cells. However, exposure to both agents resulted in a survival rate of 24.8+/−1.6 (P<0.05, Students t-test). We also compared pre- and post 5-aza samples from the same patients: in all cases 5-aza treatment increased the GO-mediated cytotoxicity from 39.4+/−3.1 to 66.8+/−2.4 ((P<0.05, Students t-test). These data show that in vivo exposure to 5-aza can induce the expression of two biomarkers involved in the response to GO. This ongoing study indicates the combination of 5-aza and GO is well-tolerated, that Syk and SHP-1 are modulated by 5-aza in vivo, and that complete responses have been noted with this combination. Disclosures: Ball: Celgene: Equity Ownership, Research Funding. Off Label Use: Will discuss use of 5-azacytidine (Vidaza) for treatment of relapsed AML in combination with Mylotarg (on label, but only as monotherapy). Medeiros:Celgene: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Merck: Research Funding; Genentech: Research Funding; Alexion: Speakers Bureau.

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Acquisition of Cytogenetic Abnormalities (CA) Is a Very Poor Prognostic Feature in Patients (pts) with Low and Intermediate-1 (int-1) Risk Myelodysplastic Syndromes (MDS),

Blood ◽

10.1182/blood.v118.21.3802.3802 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3802-3802

Author(s):

Elias Jabbour ◽

Hagop M. Kantarjian ◽

Xuemei Wang ◽

Lynne V. Abruzzo ◽

A Megan Cornelison ◽

...

Keyword(s):

Lower Risk ◽

Research Funding ◽

Regression Models ◽

Cox Model ◽

Time Dependent ◽

P Value ◽

Increased Risk ◽

History Of ◽

Previously Treated ◽

The Impact

Abstract Abstract 3802 Background and Aim: The impact of the CA on prognosis and transformation into acute myeloid leukemia among pts with low and int-1 risk MDS is not known. The aims of the study were to assess the impact of CA on the natural history of pts with lower risk MDS and to identify factors associated with its development. Methods: We reviewed 721 pts clinical records of low and intermediate risk MDS pts from 2000–2010 and conducted a retrospective analysis of all pts with at least two consecutive cytogenetic analysis (365 patients, 51%). The acquisition of CA was defined by structural change or gain in at least 2 metaphases and loss in 3 metaphases, or otherwise confirmed by FISH. Cox proportional hazards regression models were fit to assess the association between transformation-free survival (TFS) or overall survival (OS) and pt characteristics. The acquisition of CA was fitted in the Cox model as a time-dependent covariate. The association between the acquisition of CA and pt characteristics was assessed through univariate and multiple logistic regression models. Results: CA was detected in 107 pts (29%) after a median follow-up of 34 months (mos). CA was observed in a median number of 4 metaphases (range, 2–30). At diagnosis, 21% and 79% of pts who acquired CA were low-and int-1risk MDS; 50% were diploid, 22% harbored chromosome 5 /7 abnormalities. At the time of acquisition of CA, the median percentage of bone marrow blasts was 4% (range, 0% to 89%), the median WBC, hemoglobin and platelets were 3.1 × 109/L, 9.5 g/dL, and 65 × 109/L, respectively; pts were low, int-1, int-2, and high-risk MDS in 3%, 42%, 26%, 29%, respectively. The median TFS and OS were 31 (95% CI: 27– 37) and 34 (95% CI: 30 – 44) mos respectively. Assessing CA as time-dependent covariate, patients with CA had a worse TFS and OS, with a median TFS and OS of 16 and 18 mos compared to 56 and 60 mos, respectively in pts without CA. Based on the multivariable Cox model and after adjusting for effects of all other covariates, pts who had acquired CA had an increased risk of transformation (HR=1.46; p-value = 0.01) or death (HR=1.50; p-value = 0.01). By multivariate analysis, female pts with prior chemotherapy had an increased risk of developing CA (OR= 5.26; p-value <0.0001). 96 pts had history of previous malignancy treated with chemotherapy +/− radiation therapy. Of those, 34 (35%) patients acquired CA. Median time from previous chemotherapy to the acquisition of CA was 61 mos (range, 11 to 180). Pts previously treated who did not acquire CA had similar outcomes to those who had never been treated and did not develop CA, while those who did develop CA whether they were previously treated or not had worse TFS and OS. Conclusion: CA occurs at a rate of 29% of pts with lower risk MDS, more common among pts with previously treated malignancy, and has a significant impact on TFS and OS, possibly reflecting genomic instability in the natural history of MDS. Disclosures: Cortes: BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding.

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Synergistic Reactivation Of Cancer/Testis Antigens By Combination Treatment With Decitabine and Histone Deacetylase Inhibitors (Valproate, Panobinostat) In Myeloid Leukemia Cells

Blood ◽

10.1182/blood.v122.21.3763.3763 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3763-3763

Author(s):

Nadja Blagitko-Dorfs ◽

Tobias Bauer ◽

Maren Prinz ◽

Wolfram Brugger ◽

Gesine Bug ◽

...

Keyword(s):

Dna Methylation ◽

Myeloid Leukemia ◽

Histone Deacetylase Inhibitors ◽

Leukemia Cells ◽

Cancer Testis Antigens ◽

Dose Dependent ◽

Hdaci Treatment ◽

Cancer Testis

Abstract Introduction Epigenetic therapies with azanucleoside DNA hypomethylating agents, alone or in combination with histone deacetylase inhibitors (HDACi), show clinical activity in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), particularly when given at non-cytotoxic doses. They are able to reactivate epigenetically silenced genes including, among others, a number of highly immunogenic proteins dubbed Cancer/testis antigens (CTAs), predominantly the CTAs located on the X chromosome. We have previously shown that decitabine can induce expression of several CTAs, including MAGEB2 and NY-ESO-1, in myeloid cells in vitro and thereby trigger an immune response (Almstedt et al., Leuk. Res. 2010). Induction of a CTA-specific cytotoxic T cell response in vivo was reported also in AML patients treated with azacitidine and sodium valproate (VPA) and correlated with clinical response (Goodyear et al., Blood 2010). To the best of our knowledge, no data have yet been reported on the effect of combination treatment with decitabine and panobinostat or sodium valproate (VPA) on CTA reactivation in myeloid leukemia. Aim We hypothesized that by combining decitabine with HDACi we could further enhance expression of CTAs in myeloid leukemia cells and thereby boost recognition of the malignant cells by the cytotoxic T lymphocytes. Methods The myeloid cell lines U937 and Kasumi-1 were treated with decitabine alone or in combination with the HDACi VPA or panobinostat applied at non-toxic concentrations (>80% cell viability). Expression of CTAs was analyzed by RT-qPCR and Western blot after 48 hours of HDACi treatment. DNA methylation of NY-ESO-1 and MAGEB2 promoter regions was quantified by pyrosequencing. Bone marrow mononuclear cells from 19 AML patients (treated with or without VPA as add-on to decitabine in the ongoing randomized phase II DECIDER clinical trial, NCT00867672) were collected before and on day 15 of treatment, in some patients also after 2 treatment cycles. CTA mRNA expression and promoter DNA methylation were quantified as described above. Results VPA or panobinostat alone did not induce MAGEB2 or NY-ESO-1 expression in vitro. However the pretreatment of cells with decitabine prior to addition of either HDACi resulted in a synergistic dose-dependent reactivation of MAGEB2 and NY-ESO-1 on the mRNA level (confirmed for the latter on the protein level). Pyrosequencing analysis of the heavily methylated NY-ESO-1 and MAGEB2 promoters revealed, as expected, no methylation changes upon HDACi treatment, but a dose-dependent hypomethylation upon decitabine. In recently initiated in vivo studies (DECIDER trial), until now cells from 19 AML patients receiving epigenetic treatment were sequentially analyzed. Induction of MAGEB2 mRNA was observed in 9 patients (from absent to a median of 0.002 relative to GAPDH, range 0.0004-0.043), with concomitant DNA hypomethylation of the MAGEB2 promoter from median 83% pretreatment methylation (range 63%-90%) to 63% posttreatment (range 44%-74%). In 5 patients modest hypomethylation without changes in MAGEB2 expression was observed (from median pretreatment values of 89% [72%-92%] to 82% [58%-87%] posttreatment). Another 5 patients disclosed neither hypomethylation nor reexpression of MAGEB2 (results as yet blinded to treatment arm and clinical response). Conclusions Combined epigenetic treatment with the hypomethylating agent decitabine and the HDACi VPA or panobinostat synergistically induced a dose-dependent reactivation of the CTAs MAGEB2 and NY-ESO-1 in vitro, accompanied by promoter hypomethylation. First translational results of the DECIDER AML trial also indicate in vivo effects of the epigenetic treatment on CTA induction. The unmasking of CTAs to the immune system by epigenetically active drugs can increase anti-tumor immune responses, and thus has clear implications for future clinical trials combining epigenetic therapy and specific immunotherapy in myeloid neoplasia. Disclosures: No relevant conflicts of interest to declare.

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Chronic Lymphocytic Leukemia Cells with Mutated Nfkbie Are Positively Selected By Microenvironmental Signals and Display Reduced Sensitivity to Ibrutinib Treatment

Blood ◽

10.1182/blood-2021-149442 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 248-248

Author(s):

Alice Bonato ◽

Riccardo Bomben ◽

Supriya Chakraborty ◽

Giulia Felician ◽

Claudio Martines ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Pi3k Inhibitor ◽

Leukemia Cells ◽

Wild Type ◽

Clonal Composition

Abstract Inactivating mutations in NF-kB pathway genes, such as the NF-kB inhibitor NFKBIE, are among the more frequent genetic lesions in chronic lymphocytic leukemia (CLL). However, the role of these genetic lesions in CLL pathogenesis and treatment resistance is still largely unknown and requires further study in in vivo models of the disease. To this end, we generated transplantable murine leukemias with inactivating NFKBIE mutations and investigated their impact on leukemia growth and response to ibrutinib (IBR) treatment. The NFKBIE mutations were introduced by CRISPR/Cas9 editing in two recently established autoreactive leukemia lines derived from the Eμ-TCL1 murine CLL model. These cell lines proliferate spontaneously in vitro in a BCR-dependent manner, but also respond with increased proliferation to certain microenvironmental signals, such as those generated by Toll-like receptor (TLR) stimulation (Chakraborty S et al, Blood 2021). To investigate whether NFKBIE mutations can affect the proliferation of these cell lines in vitro, we performed competition experiments with mixed cultures of cells with wild type and mutated NFKBIE. Analysis of the clonal composition after 2 weeks showed no change in the mutant allele frequency (MAF), suggesting that NFKBIE mutations do not affect the spontaneous in vitro growth of the immortalized leukemia cells. However, repeated TLR or BCR stimulation of these cells with CpG-DNA, LPS, anti-IgM or autoantigen resulted in a 2-3 fold increase in MAF, suggesting that NFKBIE mutations provide a growth advantage when the cells are exposed to certain microenvironmental signals (n=3 experiments/condition, P<0.05 for each condition). To investigate the impact of NFKBIE mutations on leukemia growth in vivo, the same cells were transplanted by intraperitoneal injection in wild type mouse recipients (n=8) and the clonal composition was determined 3 weeks later by MAF analysis of cells isolated from peritoneal cavity (PC), blood and spleen. A significant increase in MAF was observed only in leukemia cells isolated from the spleen (P<0.05), suggesting that microenvironmental signals that positively select NFKBIE-mutated cells are available only in certain tissue compartments. Because mutations in other NF-kB pathway genes have been associated with resistance to IBR in mantle cell lymphoma, we next investigated whether NFKBIE mutations can also affect the response to IBR treatment. In vitro BrdU-incorporation experiments showed that IBR inhibits the proliferation of cells with mutated NFKBIE to a significantly lesser extent compared to cells with wild type NFKBIE (% proliferating cells with wild type and mutated NFKBIE, respectively, cultured without IBR: 90% vs 88%, P=n.s., with 0.2 μM IBR: 57% vs 73%, P<0.001, with 1.0 μM IBR: 28% vs 53%, P<0.001). Consistent with this finding, positive selection of NFKBIE-mutated cells was observed in the presence of IBR after 14 days in mixed culture competition experiments (mean MAF without IBR 47%, with 0.2 μM IBR 61%, p=0.032, with 1.0 μM IBR 64%, p=0.034). The greater resistance of NFKBIE-mutated cells to IBR was further validated by in vivo competition experiments showing a significantly greater increase in MAF in mice treated with IBR compared to controls in all three investigated compartments (n=4 mice/group, PC: P=0.029, blood P=0.029, spleen: P=0.001). To validate these findings in the clinical setting, we investigated the presence of NFKBIE mutations in a cohort of 84 IBR-treated CLL patients. Mutations of NFKBIE were detected at pre-treatment in 10/84 patients, 7/10 with >10% VAF values. Kaplan Meier analysis showed a trend towards reduced progression-free and overall survival from the beginning of IBR treatment for NFKBIE-mutated cases (Figure 1A). Analysis of an extended cohort of over 200 cases is ongoing and will be presented at the meeting. Finally, to investigate whether leukemic cells with mutated NFKBIE remain sensitive to other BCR inhibitors, we tested their growth in the presence of the PI3K inhibitor idelalisib or SYK inhibitor fostamatinib (Figure 1B). In contrast to IBR, both drugs inhibited the proliferation of NFKBIE-mutated cells in vitro, with a greater effect observed with idelalisib. Collectively, these data demonstrate that NFKBIE mutations can reduce the response to IBR treatment and suggest that such cases may benefit more from treatment with a PI3K inhibitor. Figure 1 Figure 1. Disclosures Marasca: Janssen: Honoraria, Other: Travel grants; AstraZeneca: Honoraria; AbbVie: Honoraria, Other: Travel grants. Tafuri: Roche: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Laurenti: Janssen: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria; BeiGene: Honoraria. Gattei: abbVie: Research Funding; Janssen: Research Funding; Menarini: Research Funding.

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DNA methylation signature of smoking in lung cancer is enriched for exposure signatures in newborn and adult blood

10.1101/356485 ◽

2018 ◽

Author(s):

K. M. Bakulski ◽

J. Dou ◽

N. Lin ◽

S. J. London ◽

J. A. Colacino

Keyword(s):

Lung Cancer ◽

Dna Methylation ◽

Lung Tumors ◽

Smoke Exposure ◽

The Cancer Genome Atlas ◽

Cigarette Smoke Exposure ◽

Aberrant Methylation ◽

Smoking During Pregnancy ◽

Never Smokers ◽

Meta Analyses

ABSTRACTBackgroundSmoking impacts DNA methylation genome-wide in blood of both newborns from maternal smoking during pregnancy and adults from personal smoking. Smoking causes lung cancer which involves aberrant methylation. We examined whether DNA methylation smoking signatures identified in blood of newborns and adults are detectable in lung tumors.MethodsWe compared smoking-related DNA methylation in lung adenocarcinomas (61 never smokers, 91 current smokers, and 238 former smokers) quantified with the Illumina450k BeadArray in The Cancer Genome Atlas with published large consortium meta-analyses of newborn and adult blood. We assessed whether CpG sites related to smoking in blood from newborns and adults were enriched in lung adenocarcinoma.ResultsTesting CpGs differentially methylated by smoke exposure (P<10−4) we identified 296 in lung tumors, while previous meta-analyses (False Discovery Rate (FDR)<0.05) identified 6,073 in newborn blood, and for adult smoking, 18,760 in blood. The lung signals were highly enriched for those seen in newborn (32 overlapping, Penrichment=1.2×10−19) and adult blood (86 overlapping, Penrichment = 9.5×10−49). The 65 genes annotated to CpGs differentially methylated in lung tumors, but not blood, were enriched for RNA processing ontologies.ConclusionsWe found highly significant overlap between smoking-related DNA methylation signals in lung cancer and those seen in blood from newborns, from in utero exposure, or adults, from their own exposure. These results suggest that some epigenetic alterations associated with cigarette smoke exposure are tissue specific, but others are common across tissues. These findings support the value of blood-based methylation biomarkers for assessing exposure effects in target tissues.

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