B cell lineage acute lymphoblastic leukemia (ALL) represents the most frequent malignancy in children and is also common in adults. Compared to patients with other malignancies, cure rates for patients with ALL are in general higher. The ALL subset with the so-called Philadelphia chromosome (Ph) encoding the oncogenic BCR- ABL1 kinase, however, has a particularly poor prognosis. Ph+ ALL is typically treated with BCR-ABL1 kinase inhibitors such as Imatinib. The treatment response to Imatinib, however, is not durable and after a latency of only a few months, Ph+ ALL cells become drug-resistant and ALL relapses. Of note, the oncogenic BCR-ABL1 kinase is not only expressed in Ph+ ALL (mainly p190 BCR-ABL1) but also in >95% of cases of chronic myeloid leukemia (CML;mainly p210 BCR-ABL1). In contrast to Ph+ ALL, long-term treatment of CML with Imatinib is effective and resistance develops only rarely. In a subgroup of patients with CML, however, the disease progresses into B lymphoid blast crisis (CML-LBC), in which treatment responses are as short-lived as in Ph+ ALL. In most cases, acquired resistance to Imatinib in Ph+ ALL and CML-LBC can be attributed to somatic mutations within the BCR-ABL1 kinase domain, which compromise the efficacy of Imatinib. In preliminary experiments for this proposal, we show that AID is specifically expressed in B cell lineage + clones of BCR-ABL1-driven leukemia (Ph ALL and CML-LBC). In these cells, AID functions as a mutator and thereby contributes to the drug-resistance typically observed in Ph+ ALL and CML-LBC. Based on these findings, our proposal addresses the question of + (1) how AID contributes to genetic instability and drug-resistance in Ph ALL (e.g. AID-specific deletions;
Aim 1), (2) to which extent AID contributes to the progression of chronic phase CML to CML-LBC (outgrowth of B lymphoid subclones that carry advantageous mutations;
Aim 2), (3) which factors cause aberrant expression of AID in Ph+ ALL and CML-LBC (Aim 3), + (4) and whether AID-expressing clones in Ph ALL and CML-LBC can be specifically targeted in a prodrug- based approach that takes advantage of the enzymatic activity of AID (Aim 4). + Together, these four Aims will help to elucidate mechanisms of drug-resistance in Ph ALL and CML-LBC and + propose a novel concept of targeted treatment Ph ALL and CML-LBC for pre-clinical evaluation.
Aim 1 : Contribution of AID to genetic instability in Ph+ ALL: We have generated BCR-ABL1-transformed B cell lineage leukemia cells with three levels of AID expression based on their genotype, namely Aid-/-, endogenous AID and forced AID-overexpression. We have injected these leukemia cells into congenic mouse recipients and will compare the developing leukemia clones by comparative genomic hybridization (CGH) analysis to identify AID-specific deletions. Deletion breakpoints will be verified by FISH analysis and mapped to AID-related somatic hypermutation hot spots. We will compare development of Imatinib-resistance in Aid-/- and Aid-wildtype leukemias developing in BCR-ABL1 p190-transgenic mice.
Aim 2 : Contribution of AID-induced mutations to progression of CML into lymphoid blast crisis: To clarify to which extent AID contributes to the progression of CML into lymphoid blast crisis, we will take two approaches. (1) Transformation of hematopoietic stem cells (HSC) by p210 BCR-ABL1 induces CML-like leukemia with subsequent progression into B lymphoid blast crisis. Studying transgenic mice expressing p210 BCR-ABL1 under control of the HSC-specific Scl-promoter on an Aid-/- background, we will investigate whether Aid-function is required for the outgrowth of B lymphoid blast crisis clones. Second, we will cross Scl-BCR-ABL1 p210 transgenic mice with an Aid-Cre reporter strain that carries YFP preceded by a loxP-flanked Stop cassette. Expression of Aid in these cells will lead to permanent genetic labeling with YFP. Based on YFP-labeling, this mouse model will indicate whether or not outgrowth of B lymphoid subclones requires expression of Aid at least at one point in time during the clonal evolution of CML Aim 3: Identification of factors that regulate AID-expression in BCR-ABL1-driven leukemias: We observed that AID expression substantially varies among primary Ph+ ALL cells from the same patient. These findings suggest that besides homogenously expressed BCR-ABL1 and B cell-specific transcription factors, additional AID-regulatory factors are only expressed in a subset of the leukemia population. Using an Aid-GFP reporter system, we will compare AIDhigh and AIDlow Ph+ ALL cells to identify key AID-regulatory factors.
Aim 4 : Prodrug-based targeting of AID-expressing Ph+ ALL cells: AID can deaminate monomeric deoxycytidine to deoxyuracil. We hypothesize that AID can likewise activate the monomeric prodrugs monomeric 5-FC (Ancobon(R)) and its derivatives 5-DFCR and Capecitabine (Xeloda(R)) into the cytotoxic metabolite 5- fluorouracil (5-FU). Taking advantage of the enzymatic activity of AID in Ph+ ALL and CML-LBC cells, we will target AID-expressing cells using 5-FC, 5-DFCR and Capecitabine for specific targeting of AID-expressing cells in Ph+ ALL and CML lymphoid blast crisis.

Public Health Relevance

The Philadelphia chromosome (Ph) encodes the oncogenic BCR-ABL1 kinase, which drives two types of leukemia: Acute lymphoblastic leukemia (Ph+ ALL) is derived from a transformed B lymphocyte and chronic myeloid leukemia (CML) originates from myeloid cells that would otherwise develop into macrophages or monocytes. While Ph+ ALL represents a rapidly progressive disease already at the outset, the course of CML is typically stable over many years and only shows rapid progression in the terminal stage, the so-called """"""""blast crisis"""""""". The reasons leading to progression from chronic phase into blast crisis are largely unknown. The treatment of both Ph+ ALL and CML has been revolutionized by the discovery of the BCR-ABL1 kinase-inhibitor Imatinib. However, even though both leukemia types carry the same genetic abnormality, the outcome of Imatinib-treatment is strikingly different: whereas Imatinib is very effective in the treatment of chronic phase CML, treatment success is only transient for patients with Ph+ ALL or blast crisis CML. In these patients, the leukemia recurs after 4 months on average and is typically drug-resistant owing to the acquisition of additional mutations. Therefore, the understanding of the underlying mutation mechanism and its potential inhibition appears to be critical for further improvement of treatment strategies of Ph+ ALL and CML. Recent work by our group demonstrated that the oncogenic BCR-ABL1 kinase in Ph+ ALL and blast crisis, but not chronic phase CML, induces expression of a mutator enzyme, termed AID (Activation-induced Cytidine Deaminase). The mutations that confer drug-resistance in Ph+ ALL and blast crisis CML can indeed be explained by activity of the AID enzyme. Additional experiments showed that engineered expression of AID in AID-negative chronic phase CML cells introduces the same mutations that cause drug-resistance in patients with AID-positive Ph+ ALL and CML blast crisis. Based on these observations, we propose four series of experiments to address the following questions: (1) Is the AID enzyme required for drug-resistance in AID-positive Ph+ ALL? To test this hypothesis, we will investigate whether BCR-ABL1-induced leukemia cells from mice carrying a deletion of the AID-gene fail to develop drug-resistance. (2) Does the AID enzyme play a critical role in the progression of chronic phase CML into blast crisis? Chronic phase CML can be treated very successfully for many years, whereas blast crisis represents a final and often fatal stage of the disease. (3) Which are the factors that induce aberrant expression of the AID enzyme in Ph+ ALL and blast crisis CML? The identification of such factors will likely help to understand how expression of this deleterious mutator enzyme can be prevented. (4) Is it possible to target AID-expressing cells by taking advantage of the enzymatic activity of AID? For this approach, we propose to use a precursor-drug that has no effect as such but will become toxic upon AID-mediated conversion. Given that AID-expressing cells are more likely to be drug-resistant than others, we propose a treatment approach that is focused on the AID-expressing leukemia cells.

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National Cancer Institute (NCI)
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Hematopoiesis Study Section (HP)
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Howcroft, Thomas K
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Children's Hospital of Los Angeles
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