Acute myeloid leukemia : apoptotic signalling and gene expression associated with treatment response

Acute myeloid leukemia (AML) is a severe, life threatening malignancy characterized by a clonal expansion of immature myeloid cells in the bone marrow, resulting in severe infections and bleedings. High dose chemotherapy is able to normalize the blood and bone marrow morphology (complete remission, CR) in a majority of treated patients, but recurrent disease, typically occurs within 1-2 years. Since further intensification of chemotherapeutic regimens is usually ineffective and accompanied by excess toxicity, novel approaches using better-targeted drugs are now being assessed. We have analysed the effects of one such new agent, gemtuzumab ozogamicin (GO) on AML cells and have also looked for biomarkers of clinical response and the role of multidrug resistance (MDR) expression utilizing biobanked cells from an AML cohort with known long-term therapuetic outcome. In paper I we analysed apoptotic signalling in response to GO, a monoclonal CD33 antibody conjugated to the DNA-double strand break-inducing toxin calicheamicin. The CD33 antigen is typically expressed on AML blast cells, but not on e.g. normal gut cells. We found that GO could induce mitochondrial depolarisation, activation of caspase-3 and decreased viability of primary cells from AML patients and AML cell lines. Moreover, we showed that GO activated the proapoptotic proteins Bak and Bax, regulators of mitochondria-mediated apoptotic signalling. Importantly, none of the above events could be observed in GO-resistant AML cells. In paper II, we looked at the role of caspase-2 in GO- or daunorubicin-induced apoptotic signalling. We noted that both drugs caused cleavage of caspase-2 into its active form. A selective caspase-2 inhibitor prevented GO-induced caspase-3 activation, yet did not influence the activation of Bak and Bax. All in all, our data indicate that both mitochondria-dependent and independent routes to caspase-3 activation are involved in GO-induced apoptotic signalling, findings that may lead to novel future therapeutic approaches for AML. Improved predictive biomarkers for treatment response are clearly needed to enable more personalized and effective therapeutic options in AML. In paper III we studied peripheral blood cells from 42 patients diagnosed with AML and subjected to induction chemotherapy, aiming to identify biomarkers of CR duration using global gene expression analysis (Affymetrix®). Prominent differences in gene expression were found with a remarkable up-regulation of the transcription factor RUNX1T1 in patients with short vs. those with long subsequent CR duration. Network analyses (Oncomine®) revealed multiple transcription factors as interactors to RUNX1T1, out of which TCF3 was also significantly up-regulated in patients with short CR duration. An in silico validation, taking advantage of previously published data from two other independent AML cohorts revealed 52 genes to be regulated in all three cohorts. Among these genes CXCL3, ZMIZ1 and PRDX2 attracted a special interest due to their reported involvement in cancer, leukemia, apoptosis and proliferation. Thus, CXCL3 and ZMIZ1, with known involvement in tumorgenesis, had increased expression in poor responders whereas PRDX2, a tumour suppressor gene, instead showed a decreased expression. In paper IV we investigated the clinical relevance of 380 genes, reported to have a role in multidrug resistance (MDR) and analyzed 11 paired sampled from AML patients, collected at diagnosis and at time of relapse. Unsupervised hierarchical clustering showed that half of the cases had a similar expression pattern at both time points, whereas in the remaining patients the MDR genes became altered, suggesting clonal evolution. Patient-by-patient analyses showed signs of unique individual patient gene signatures and in 10 out of 11 patients an increase of at least one ABC transporter was observed at relapse. These findings call for a more broad signalling analysis of diagnostic and relapse AML blasts in order to improve chemotherapy response and thereby overall survival of the individual AML patient

Predictive value of DNA methylation patterns in AML patients treated with an azacytidine containing induction regimen

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease with a poor prognosis. Dysregulation of the epigenetic machinery is a significant contributor to disease development. Some AML patients benefit from treatment with hypomethylating agents (HMAs), but no predictive biomarkers for therapy response exist. Here, we investigated whether unbiased genome-wide assessment of pre-treatment DNA-methylation profiles in AML bone marrow blasts can help to identify patients who will achieve a remission after an azacytidine-containing induction regimen. RESULTS: A total of n = 155 patients with newly diagnosed AML treated in the AMLSG 12-09 trial were randomly assigned to a screening and a refinement and validation cohort. The cohorts were divided according to azacytidine-containing induction regimens and response status. Methylation status was assessed for 664,227 500-bp-regions using methyl-CpG immunoprecipitation-seq, resulting in 1755 differentially methylated regions (DMRs). Top regions were distilled and included genes such as WNT10A and GATA3. 80% of regions identified as a hit were represented on HumanMethlyation 450k Bead Chips. Quantitative methylation analysis confirmed 90% of these regions (36 of 40 DMRs). A classifier was trained using penalized logistic regression and fivefold cross validation containing 17 CpGs. Validation based on mass spectra generated by MALDI-TOF failed (AUC 0.59). However, discriminative ability was maintained by adding neighboring CpGs. A recomposed classifier with 12 CpGs resulted in an AUC of 0.77. When evaluated in the non-azacytidine containing group, the AUC was 0.76. CONCLUSIONS: Our analysis evaluated the value of a whole genome methyl-CpG screening assay for the identification of informative methylation changes. We also compared the informative content and discriminatory power of regions and single CpGs for predicting response to therapy. The relevance of the identified DMRs is supported by their association with key regulatory processes of oncogenic transformation and support the idea of relevant DMRs being enriched at distinct loci rather than evenly distribution across the genome. Predictive response to therapy could be established but lacked specificity for treatment with azacytidine. Our results suggest that a predictive epigenotype carries its methylation information at a complex, genome-wide level, that is confined to regions, rather than to single CpGs. With increasing application of combinatorial regimens, response prediction may become even more complicated

Molecular stratification and residual disease detection in acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease with a variable response totreatment. Although the majority of AML patients achieve a complete morphologic remission after induction chemotherapy, relapse remains the major cause of death in AML. The objective of this thesis was to further refine risk classification at diagnosis and investigate the value of molecular residual disease detection by next-generation sequencing during treatment, with the ultimate goal to improve relapse prediction in AML.<br/

Molecular stratification and residual disease detection in acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease with a variable response totreatment. Although the majority of AML patients achieve a complete morphologic remission after induction chemotherapy, relapse remains the major cause of death in AML. The objective of this thesis was to further refine risk classification at diagnosis and investigate the value of molecular residual disease detection by next-generation sequencing during treatment, with the ultimate goal to improve relapse prediction in AML.<br/

Myeloid Leukemia

This book comprises a series of chapters from experts in the field of diagnosis and treatment of myeloid leukemias from all over the world, including America, Europe, Africa and Asia. It contains both reviews on clinical aspects of acute (AML) and chronic myeloid leukemias (CML) and original publications covering specific clinical aspects of these important diseases. Covering the specifics of myeloid leukemia epidemiology, diagnosis, risk stratification and management by authors from different parts of the world, this book will be of interest to experienced hematologists as well as physicians in training and students from all around the globe

Prognostic Implications of Cellular Senescence in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease for which biologically grounded predictors of outcome remain a clinical need. In addition to tumor cell death, antineoplastic drugs can mediate a long-lasting growth arrest of vital, metabolically active tumor cells termed therapy-induced senescence, but structured investigations into its prognostic and predictive power are lacking. Besides its occurrence in response to chemotherapeutic treatment, cellular senescence can be evoked by replicative stress or activation of oncogenes and serves as an initial barrier to cancer development. Yet, long-term effects of senescent cancer cells on tumor growth are unclear as they are known to mediate inflammation via a senescence-associated secretory phenotype (SASP) and are subjected to epigenetic remodeling, thereby acquiring cancer stemness characteristics. In an ex vivo analysis of AML blast samples from patients at diagnosis, I aimed to characterize basal as well as treatment-evoked senescence and determine its role as a prognostic and predictive biomarker. I established assays to detect and therapeutically induce senescence in a primary AML culture setting. Senescence was assessed by senescence-associated β-galactosidase (SA-β-gal) activity and other senescence markers. Gene expression analyses validated my experimental characterization of AML samples as “senescent”, as evidenced by upregulation of senescence-associated gene expression signatures. For prognostic analysis, clinical outcomes and molecular genetics of AML sample donors were retrieved. I found the intra-individual changes of senescence levels in response to the standard anti-leukemic agent daunorubicin to be positively correlated with better disease-free- and overall patient survival. In line with this, a more favorable molecular risk group, normal karyotype, and NPM1 as well as DNMT3AR882 mutations were associated with higher therapy-induced senescence levels. Other therapeutic AML agents, namely hydroxyurea, decitabine and gemtuzumab ozogamicin were also shown to induce senescence. Finally, in a consecutive ex vivo treatment with daunorubicin (to induce senescence), followed by the “senolytic” (i.e., selectively cytotoxic to senescent cells) BCL2 inhibitors venetoclax and navitoclax, both growth and viability of AML blasts were additionally reduced compared to single-agent treatments only in senescence-capable samples. To the best of my knowledge, this is the first study providing direct evidence that cellular senescence, induced ex vivo in patient-derived AML blasts by chemotherapeutic drugs, could serve as a predictive biomarker of long-term response to standard therapy. I believe that therapy-induced senescence might explain, at least in part, the underlying biology of current paraclinical risk indicators, and, as an outlook, might serve as a guidance for future personalized treatment of AML.Die akute myeloische Leukämie (AML) ist eine heterogene Erkrankung, für die die Entwicklung neuer pathophysiologisch fundierter prädiktiver Biomarker von großer klinischer Notwendigkeit ist. Zusätzlich zu apoptotischem Zelltod von Tumorzellen können antineoplastische Medikamente zu einem dauerhaften Zellzyklusarrest viabler, metabolisch aktiver Tumorzellen führen, welches Phänomen als Therapie-induzierte Seneszenz in verschiedenen Tumorentitäten charakterisiert wurde. Die prognostische und prädiktive Relevanz Therapie-induzierter Seneszenz für den Verlauf von Tumorerkrankungen ist derzeit unklar. Außer einer Induktion durch Chemotherapeutika kann Seneszenz u.a. durch replikativen Stress oder Onkogen-Expression hervorgerufen werden und dient dadurch als initiale zelluläre Barriere gegen maligne Entartung. Die langfristige Bedeutung von im Organismus persistierenden seneszenten Tumorzellen bleibt jedoch unklar, da diese durch ihren Seneszenz-assoziierten sekretorischen Phänotyp (SASP) auch proinflammatorisch wirken und durch epigenetische Veränderungen Krebsstammzelleigenschaften aufweisen können. In ex vivo-Untersuchungen an aus Patient:innenproben zum Zeitpunkt der Diagnosestellung gewonnenen AML-Blasten konnte ich zunächst „basale“ und Therapie-bedingte Seneszenz in der AML charakterisieren um daraufhin Seneszenz als prädiktiven Biomarker zu analysieren. Nach Etablierung von Primärkulturbedingungen für die zytostatische Behandlung (und somit mögliche Seneszenzinduktion) aufgereinigter AML-Blasten konnte ich mit zytochemischen und Fluoreszenz-basierten Assays die Zunahme der Seneszenz-assoziierten-β-Galaktosidase (SA-β-gal)-Aktivität und anderer Seneszenzmarker nachweisen. Durch RNA-Sequenzierung konnte meine experimentelle Klassifikation individueller AML-Proben als „Seneszenz-fähig“ anhand Seneszenz-assoziierter Genexpressionssignaturen bestätigt und weiter charakterisiert werden. Zur Analyse der prädiktiven Bedeutung Therapie-bedingter Seneszenz wurden die einzelnen AML Proben weiter molekulargenetisch untersucht und experimentelle Ergebnisse mit dem jeweiligen klinischen Verlauf individueller Patient:innen korreliert. Ich konnte zeigen, dass die intraindividuelle Induzierbarkeit von Seneszenz durch ex vivo-Behandlung mit dem AML-Standardchemotherapeutikum Daunorubicin positiv mit einem verbesserten erkrankungsfreien Überleben und Gesamtüberleben korrelierte. Zudem waren eine günstigere molekulare Risikogruppe, ein normaler Karyotyp sowie NPM1- und DNMT3AR882-Mutationen mit höheren Leveln Therapie-induzierter Seneszenz assoziiert. Durch die Behandlung mit anderen AML-Therapeutika wie Hydroxyurea, Decitabin oder Gemtuzumab-Ozogamicin konnte ebenfalls Seneszenz ausgelöst werden. Schließlich konnten ich durch eine konsekutive ex vivo-Behandlung mit zunächst Daunorubicin (zur Seneszenzinduktion) und darauffolgend mit den „senolytisch“ wirkenden (d.h. selektiv zytotoxisch gegenüber seneszenten Zellen) BCL2-Inhibitoren Venetoclax und Navitoclax sowohl Zellzahl als auch Viabilität seneszenzfähiger AML-Proben im Vergleich zu einer Therapie mit den Einzelsubstanzen oder zu AML-Proben, welche nicht seneszenzfähig waren, zusätzlich reduzieren. Nach meinem Kenntnisstand konnte im Rahmen dieses Promotionsprojektes erstmals nachgewiesen werden, dass durch ex vivo-Chemotherapie in aus Patient:innen gewonnenen AML-Blasten induzierte zelluläre Seneszenz als prädiktiver Biomarker für das langzeitige Therapieansprechen auf die Standard-Induktionstherapie dienen kann. Möglicherweise erklärt Therapie-induzierte Seneszenz Teilaspekte der etablierten paraklinischen Risikofaktoren zugrundliegenden Tumorbiologie und kann perspektivisch als Marker für personalisierte Behandlungskonzepte in der AML verwendet werden

Multiplex Screening for Interacting Compounds in Paediatric Acute Myeloid Leukaemia

Paediatric acute myeloid leukaemia (AML) is a heterogeneous disease characterised by the malignant transformation of myeloid precursor cells with impaired differentiation. Standard therapy for paediatric AML has remained largely unchanged for over four decades and, combined with inadequate understanding of the biology of paediatric AML, has limited the progress of targeted therapies in this cohort. In recent years, the search for novel targets for the treatment of paediatric AML has accelerated in parallel with advanced genomic technologies which explore the mutational and transcriptional landscape of this disease. Exploiting the large combinatorial space of existing drugs provides an untapped resource for the identification of potential combination therapies for the treatment of paediatric AML. We have previously designed a multiplex screening strategy known as Multiplex Screening for Interacting Compounds in AML (MuSICAL); using an algorithm designed in-house, we screened all pairings of 384 FDA-approved compounds in less than 4000 wells by pooling drugs into 10 compounds per well. This approach maximised the probability of identifying new compound combinations with therapeutic potential while minimising cost, replication and redundancy. This screening strategy identified the triple combination of glimepiride, a sulfonylurea; pancuronium dibromide, a neuromuscular blocking agent; and vinblastine sulfate, a vinca alkaloid, as a potential therapy for paediatric AML. We envision that this approach can be used for a variety of disease-relevant screens allowing the efficient repurposing of drugs that can be rapidly moved into the clinic