Genomes and Genes
AID-mediated genetic instability in BCR-ABL1-transformed B cell lineage leukemia
Principal Investigator: MARKUS MUSCHEN
Affiliation: University of Southern California
Abstract: DESCRIPTION (provided by applicant): 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.
Funding Period: 2009-03-18 - 2014-01-31
more information: NIH RePORT
- Human chromosomal translocations at CpG sites and a theoretical basis for their lineage and stage specificityAlbert G Tsai
Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, MC9176, Los Angeles, CA 90089 9176, USA
Cell 135:1130-42. 2008....
- SOX4 enables oncogenic survival signals in acute lymphoblastic leukemiaParham Ramezani-Rad
Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143, USA
Blood 121:148-55. 2013..Collectively, these studies identify SOX4 as a central mediator of oncogenic PI3K/AKT and MAPK signaling in ALL...
- Integrin alpha4 blockade sensitizes drug resistant pre-B acute lymphoblastic leukemia to chemotherapyYao Te Hsieh
Department of Pediatrics, Division of Hematology and Oncology, Children s Hospital Los Angeles, University of Southern California Keck School of Medicine, Los Angeles, CA 90027, USA
Blood 121:1814-8. 2013..Chemotherapy combined with Natalizumab prolonged survival of NOD/SCID recipients of primary ALL, suggesting adjuvant alpha4 inhibition as a novel strategy for pre-B ALL...
- O-acetylated N-acetylneuraminic acid as a novel target for therapy in human pre-B acute lymphoblastic leukemiaReshmi Parameswaran
Section of Molecular Carcinogenesis, Division of Hematology Oncology, The Saban Research Institute, Children s Hospital Los Angeles, CA 90089, USA
J Exp Med 210:805-19. 2013..This demonstrates that Neu5Ac 9-O-acetylation is essential for survival of these cells and suggests that Neu5Ac de-O-acetylation could be used as therapy to eradicate drug-resistant ALL cells...
- Small-molecule inhibition of CBP/catenin interactions eliminates drug-resistant clones in acute lymphoblastic leukemiaE J Gang
Children s Hospital Los Angeles, Division of Hematology and Oncology, Department of Pediatrics, University of Southern California, Keck School of Medicine, Los Angeles, CA, USA
Oncogene 33:2169-78. 2014..Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL. ..
- Personalized synthetic lethality induced by targeting RAD52 in leukemias identified by gene mutation and expression profileKimberly Cramer-Morales
Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140, USA
Blood 122:1293-304. 2013..We believe this work may initiate a personalized therapeutic approach in numerous patients with tumors displaying encoded and functional BRCA deficiency. ..
- BACH2 mediates negative selection and p53-dependent tumor suppression at the pre-B cell receptor checkpointSrividya Swaminathan
Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA
Nat Med 19:1014-22. 2013..These findings identify BACH2 as a crucial mediator of negative selection at the pre-B cell receptor checkpoint and a safeguard against leukemogenesis. ..
- BACH2-BCL6 balance regulates selection at the pre-B cell receptor checkpointSrividya Swaminathan
Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA 94143, USA
Trends Immunol 35:131-7. 2014..Here, we discuss the antagonism between BCL6 and BACH2 during early B cell development, as well as its implications in both repertoire selection and counter-selection of premalignant clones for leukemia suppression...
- AID downregulation is a novel function of the DNMT inhibitor 5-aza-deoxycytidineChiou Tsun Tsai
Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan
Oncotarget 5:211-23. 2014..The study not only displays the association of AID and DNMT1 and identifies a novel biological function of AID, but also provides novel information regarding the use of DNMT inhibitors to treat AID-positive hematopoietic cancers. ..
- Targeting the B-cell receptor signaling pathway in B lymphoid malignanciesMaike Buchner
Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California, USA
Curr Opin Hematol 21:341-9. 2014..Here, we summarize the recent experimental evidence for the importance of BCR signaling and clinical concepts to target the BCR pathway in B-cell leukemia and lymphoma...
- Diet-induced obesity accelerates acute lymphoblastic leukemia progression in two murine modelsJason P Yun
Division of Endocrinology, Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA
Cancer Prev Res (Phila) 3:1259-64. 2010..This is the first study to show that obesity can directly accelerate the progression of ALL. Thus, the observed associations between obesity and leukemia incidence are likely to be directly related to biological effects of obesity...
- SNP array analysis of tyrosine kinase inhibitor-resistant chronic myeloid leukemia identifies heterogeneous secondary genomic alterationsDaniel Nowak
Division of Hematology and Oncology, Cedars Sinai Medical Center, University of California Los Angeles School of Medicine, CA 90048, USA
Blood 115:1049-53. 2010..This may support a hypothesis of TKI-induced selection of subclones differentiating into immature B-cell progenitors as a mechanism of disease progression and evasion of TKI sensitivity...
- Identification and functional relevance of de novo DNA methylation in cancerous B-cell populationsXiao Ming Wang
Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles, California 90089, USA
J Cell Biochem 109:818-27. 2010..Furthermore, we propose that similar to de novo genetic mutations, the majority of de novo DNA methylation events observed in tumors are passengers not causally involved in tumorigenesis...
- Development of resistance to dasatinib in Bcr/Abl-positive acute lymphoblastic leukemiaF Fei
Section of Molecular Carcinogenesis, Division of Hematology Oncology, The Saban Research Institute of Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA
Leukemia 24:813-20. 2010....
- A functional receptor for B-cell-activating factor is expressed on human acute lymphoblastic leukemiasReshmi Parameswaran
Section of Molecular Carcinogenesis, Division of Hematology Oncology, Childrens Hospital Los Angeles, CA 90027, USA
Cancer Res 70:4346-56. 2010..Our report is the first showing universal expression of BAFF-R by pre-B ALL cells and opens the possibility of blocking its function as an adjuvant therapeutic strategy...
- BCL6-mediated repression of p53 is critical for leukemia stem cell survival in chronic myeloid leukemiaChristian Hurtz
Department of Laboratory Medicine, University of California San Francisco, CA 94143, USA
J Exp Med 208:2163-74. 2011..Clinical validation of this concept could limit the duration of TKI treatment in CML patients, which is currently life-long, and substantially decrease the risk of blast crisis transformation...
- Pre-B cell receptor-mediated activation of BCL6 induces pre-B cell quiescence through transcriptional repression of MYCRahul Nahar
Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA
Blood 118:4174-8. 2011..Hence, pre-B cell receptor-mediated activation of BCL6 limits pre-B cell proliferation and induces cellular quiescence at the small pre-BII (Fraction D) stage...
- Integrated microfluidic and imaging platform for a kinase activity radioassay to analyze minute patient cancer samplesCong Fang
Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, University of California, Los Angeles, California 90095, USA
Cancer Res 70:8299-308. 2010..The low sample input requirement of the device creates new potential for direct kinase activity experimentation and diagnostics on patient blood, bone marrow, and needle biopsy samples...
- Activation-induced cytidine deaminase accelerates clonal evolution in BCR-ABL1-driven B-cell lineage acute lymphoblastic leukemiaTanja Andrea Gruber
Childrens Hospital Los Angeles and Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA
Cancer Res 70:7411-20. 2010..We conclude that AID accelerates clonal evolution in BCR-ABL1 ALL by enhancing genetic instability and aberrant somatic hypermutation, and by negative regulation of tumor-suppressor genes...
- BCL6 is critical for the development of a diverse primary B cell repertoireCihangir Duy
Childrens Hospital Los Angeles and Leukemia and Lymphoma Program, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90027, USA
J Exp Med 207:1209-21. 2010..We conclude that negative regulation of Arf by BCL6 is required for pre-B cell self-renewal and the formation of a diverse polyclonal B cell repertoire...
- BCL6 enables Ph+ acute lymphoblastic leukaemia cells to survive BCR-ABL1 kinase inhibitionCihangir Duy
Department of Laboratory Medicine, University of California San Francisco, San Francisco, California 94143, USA
Nature 473:384-8. 2011..We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones...
- Targeting survivin overcomes drug resistance in acute lymphoblastic leukemiaEugene Park
Department of Pediatrics, Division of Hematology and Oncology, Children s Hospital Los Angeles, University of Southern California Keck School of Medicine, Los Angeles, CA, USA
Blood 118:2191-9. 2011..These findings show the importance of survivin expression in drug resistance and demonstrate that survivin inhibition may represent a powerful approach to overcoming drug resistance and preventing relapse in patients with ALL...
- Global phosphoproteomics reveals crosstalk between Bcr-Abl and negative feedback mechanisms controlling Src signalingLiudmilla Rubbi
Crump Institute for Molecular Imaging, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA
Sci Signal 4:ra18. 2011..The identified Bcr-Abl-activated SFK regulatory mechanisms are candidates for dysregulation during leukemia progression and acquisition of SFK-mediated drug resistance...
- Pre-B cell receptor signaling in acute lymphoblastic leukemiaRahul Nahar
Leukemia and Lymphoma Program, USC Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA, USA
Cell Cycle 8:3874-7. 2009....
- The B cell mutator AID promotes B lymphoid blast crisis and drug resistance in chronic myeloid leukemiaLars Klemm
Leukemia and Lymphoma Program, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90027, USA
Cancer Cell 16:232-45. 2009..Importantly, our data uncover a causative role of AID activity in the acquisition of BCR-ABL1 mutations leading to Imatinib resistance, thus providing a rationale for the rapid development of drug resistance and blast crisis progression...
- Pre-B cell receptor-mediated cell cycle arrest in Philadelphia chromosome-positive acute lymphoblastic leukemia requires IKAROS functionDaniel Trageser
Leukemia and Lymphoma Program, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90027, USA
J Exp Med 206:1739-53. 2009....