Lineage tracing of acute myeloid leukemia reveals the impact of hypomethylating agents on chemoresistance selection

Chemotherapy-resistant cancer recurrence is a major cause of mortality. In acute myeloid leukemia (AML), chemorefractory relapses result from the complex interplay between altered genetic, epigenetic and transcriptional states in leukemic cells. Here, we develop an experimental model system using in vitro lineage tracing coupled with exome, transcriptome and in vivo functional readouts to assess the AML population dynamics and associated molecular determinants underpinning chemoresistance development. We find that combining standard chemotherapeutic regimens with low doses of DNA methyltransferase inhibitors (DNMTi, hypomethylating drugs) prevents chemoresistant relapses. Mechanistically, DNMTi suppresses the outgrowth of a pre-determined set of chemoresistant AML clones with stemness properties, instead favoring the expansion of rarer and unfit chemosensitive clones. Importantly, we confirm the capacity of DNMTi combination to suppress stemness-dependent chemoresistance development in xenotransplantation models and primary AML patient samples. Together, these results support the potential of DNMTi combination treatment to circumvent the development of chemorefractory AML relapses

Disseção quantitativa da dinâmica subclonal subjacente à recidiva de leucemia mielóide aguda

Tese de mestrado, Oncobiologia, Universidade de Lisboa, Faculdade de Medicina, 2017Na mesma linha dos estudos da especiação Darwinista, os tumores geralmente surgem através da evolução ramificada de uma única linhagem celular inicialmente transformada (clone fundador). Apesar de inicialmente se pensar que os tumores consistiam numa população celular homogénea, múltiplos estudos têm demonstrado que existe uma profunda heterogeneidade molecular intratumoral em cada cancro, onde coexistem múltiplos subclones. A aplicação de terapia conduz à seleção de subclones pré-existentes, que sinergiza com mutações de novo, gerando mais heterogeneidade intratumoral e o aparecimento de variantes resistentes à terapêutica, que acabam por originar recidiva. Na realidade, a heterogeneidade intratumoral observada em cada doente constitui a maior barreira ao tratamento e cura do cancro. A leucemia mielóide aguda (LMA) é uma doença maligna hematológica heterogénea e agressiva, caraterizada pelo bloqueio da diferenciação mielóide e pela proliferação descontrolada das células mielóides progenitoras transformadas. LMA é o tipo de cancro hematológico mais mortal e a leucemia aguda mais frequente. A quimioterapia induz remissões completas na maioria dos doentes (50-75%) mas muitos têm recidiva com clones resistentes e destes apenas 10% sobrevivem. As alterações genéticas que influenciam o prognóstico em LMA estão bem caraterizadas. No entanto, os mecanismos moleculares que interferem ao nível subclonal, assim como a natureza do fenómeno da recorrência à terapia convencional, não são claros, principalmente devido ao facto de faltarem sistemas experimentais que nos permitam estudar variantes subclonais que se tornam resistentes após terapia, particularmente com a resolução de linhagens de células únicas. Esta tese teve como objetivo dissecar a dinâmica subclonal responsável pela recidiva em LMA. Em detalhe, clarificar a natureza do processo de recidiva em LMA após tratamento com quimioterapia convencional (citarabina combinada com antraciclinas) e com terapias emergentes (decitabina), de modo a esclarecer como diferentes terapias interferem com o desenvolvimento de linhagens/subclones resistentes às mesmas e determinar se estas terapias resultam num processo de recidiva estocástico ou pré-determinado. Posteriormente, quisemos também verificar se os resultados obtidos eram consistentes entre diferentes subtipos de LMA. Utilizou-se uma tecnologia de marcação celular que permite estudar a dinâmica subclonal, através do rastreio em grande escala de várias linhagens celulares em paralelo. Esta tecnologia permite a incorporação de códigos de barras celulares (sequências únicas de DNA não codificante, rastreáveis por SNG) no genoma de células individuais de LMA. Assim, foi possível seguir os descendentes de todas as células de LMA submetidas a terapia e que sobreviveram à mesma, através da análise quantitativa baseada em SNG das frequências dos diferentes códigos de barras moleculares, permitindo perceber se o processo de recidiva após terapêutica foi estocástico (diferentes distribuições de códigos de barras na recidiva em replicados tratados com a mesma terapia) ou determinístico (distribuições de códigos de barras semelhantes na recidiva em replicados tratados com a mesma terapia). Adicionalmente, realizaram-se análises funcionais, que permitiram avaliar a cinética de crescimento de células submetidas a terapia ao longo do tempo, de modo a verificar se as células sobreviventes à terapia eram resistentes ou sensíveis à mesma. Os resultados obtidos nesta tese mostraram que a terapia originou alterações na arquitetura dos códigos de barras apenas após ser removida. Apesar de haver uma seleção de subclones específicos nas amostras tratadas quer com quimioterapia convencional quer com a combinação de quimioterapia convencional e decitabina, os resultados mostraram que a adição de decitabina à quimioterapia convencional resultou num aumento da estocasticidade do processo de recidiva, nos casos em que a quimioterapia foi responsável por uma grande pressão seletiva. Para além disto, os resultados mostraram também que o tratamento com quimioterapia convencional selecionou subclones resistentes à terapia, enquanto os subclones sobreviventes após tratamento com combinação de quimioterapia convencional e decitabina não adquiriram resistência. Não foi, no entanto, possível concluir se este fenómeno era transversal a vários subtipos de LMA devido a diferenças na suscetibilidade aos tratamentos, e decorrente variabilidade na pressão seletiva aplicada. Concluindo, estes resultados sugerem que a quimioterapia convencional afeta o potencial de crescimento de cada linhagem diferencialmente, contribuindo para originar resistência às terapias. A adição de decitabina, quando acompanhada de alta pressão seletiva e consequente indução de maior estocasticidade do processo de recidiva, reverte a resistência observada nas amostras submetidas a tratamento com quimioterapia convencional, pelo que devia ser ponderada a combinação de quimioterapia e decitabina como terapia de primeira linha. No futuro, para percebermos melhor o fenómeno de resistência em LMA, pretende-se dissecar os mecanismos moleculares que interferem na resistência de leucemia mielóide aguda, ao nível da genómica e transcritómica.In the same line as studies of Darwinian speciation, tumors usually arise through branched evolution of a single transformed lineage (the founding clone). Although tumors were initially thought to consist of a homogeneous population, multiple studies have shown that there is a profund intratumoral molecular heterogeneity in each cancer, with the coexistence of multiple subclones. Therapy leads to the selection of preexisting subclones, which synergizes with de novo mutations, leading to more intratumor heterogeneity and the emergence of therapeutically resistant variants that end up causing recurrence. In fact, the intratumor heterogeneity observed in each patient constitutes the greatest barrier to treatment. Acute myeloid leukemia (AML) is a heterogeneous and aggressive hematological malignancy characterized by the blockage of myeloid differentiation and the uncontrolled proliferation of the transformed myeloid progenitor cells. AML is the most lethal hematological cancer and the most common acute leukemia. Chemotherapy induces complete remissions in most patients (50-75%) but many relapse with resistant clones and of these only 10% survive. Genetic changes that influence the prognosis in AML have been characterized. However, the molecular mechanisms that interfere at the subclonal level, as well as the nature of the recurrence phenomenon, have not yet been discovered. This is mainly due to the lack of experimental systems that allow us to study subclonal variants which become resistant after standard therapy, in particular with the resolution of single-cell lineages. This thesis aimed to dissect the subclonal dynamics responsible for recurrence in AML and to study the nature of the recurrence process in AML after treatment with first-line chemotherapy (cytarabine combined with anthracyclines) and with emerging therapies (decitabine). Aditionally, we aimed to clarify how different therapies interfere with the development of resistant clonal populations and determine if these therapies result in a stochastic or predetermined recurrence process. Subsequently, we also wanted to verify if the results were consistent between different AML subtypes. We used a lentiviral-based cellular barcoding technology, allowing us to study subclonal dynamics through large-scale screening of several cell lineages in parallel. This technology allows the incorporation of cellular barcodes (unique sequences of non-coding DNA, traceable by NGS) into the genome of individual AML cells. Thus, it was possible to follow the descendants of all surviving AML cells through the NGS-based quantitative analysis of the frequencies of the different molecular barcodes in the populations resistant to therapy, allowing us to see if the recurrence process was stochastic (different distributions of barcodes upon relapse in replicates treated with the same therapy) or deterministic (similar distributions of barcodes upon relapse in replicates treated with the same therapy). In addition, functional analyzes were carried out to evaluate the growth kinetics of treated cells over time (in order to verify if the cells surviving the therapy were resistant to the therapy). The results obtained in this thesis showed that the therapy originated changes in the architecture of barcodes only after the remotion of the therapy. Although there was a selection of specific subclones in the treated samples either with conventional chemotherapy or with combination of conventional chemotherapy and decitabine, the results showed that the addition of decitabine to conventional chemotherapy resulted in an increase of stochacity in the relapse process, in the cases where chemotherapy was responsible for strongest selective pressure. In addition, the results also showed that treatment with conventional chemotherapy gave rise to subclones resistant to therapy, while surviving subclones after treatment with a combination of conventional chemotherapy and decitabine were not resistant. However, we could not conclude whether this was true across various AML subtypes due to differences in susceptibility to treatment, thus resulting in variability in the selective pressure applied. In conclusion, these results suggest that standard chemotherapy affects the growth potential of each lineage differentially, thus contributing to resistance to therapies. The addition of decitabine, when accompanied by high selective pressure and consequent induction of a greater stochasticity of the relapse process, reverses the resistance observed in the samples submitted to treatment with conventional chemotherapy, so the combination of chemotherapy and decitabine should be considered as first-line therapy. In the future, to better understand the phenomenon of resistance in AML, we intend to dissect the molecular mechanisms that interfere in the resistance of acute myeloid leukemia, at the levels of genomics and transcriptomics

Author Correction: Lineage tracing of acute myeloid leukemia reveals the impact of hypomethylating agents on chemoresistance selection

An amendment to this paper has been published and can be accessed via a link at the top of the pape Chemotherapy-resistant cancer recurrence is a major cause of mortality. In acute myeloid leukemia (AML), chemorefractory relapses result from the complex interplay between altered genetic, epigenetic and transcriptional states in leukemic cells. Here, we develop an experimental model system using in vitro lineage tracing coupled with exome, transcriptome and in vivo functional readouts to assess the AML population dynamics and associated molecular determinants underpinning chemoresistance development. We find that combining standard chemotherapeutic regimens with low doses of DNA methyltransferase inhibitors (DNMTi, hypomethylating drugs) prevents chemoresistant relapses. Mechanistically, DNMTi suppresses the outgrowth of a pre-determined set of chemoresistant AML clones with stemness properties, instead favoring the expansion of rarer and unfit chemosensitive clones. Importantly, we confirm the capacity of DNMTi combination to suppress stemness-dependent chemoresistance development in xenotransplantation models and primary AML patient samples. Together, these results support the potential of DNMTi combination treatment to circumvent the development of chemorefractory AML relapses