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Acute myeloid leukemia therapeutic target deconvolution by compound clustering and synergistic fingerprinting
Short Talk Presentations/ Experimental Hematology 44 (2016) S40–S54
missense mutations in MECOM, encoding EVI1. Subsequent analysis of MECOM in two other individuals with ATRUS revealed two additional missense mutations. These three mutations (Thr756Ala, His751Arg, Arg750Trp) were clustered within the 8th zinc finger motif of the C-terminal zinc finger domain of EVI1. Chromatin immunoprecipitation and quantitative polymerase chain reaction assays of the regions harboring the ETS-like motif that is known as an EVI1 binding site showed a reduction in immunoprecipitated DNA for two EVI1 mutants compared with wild-type EVI1. Furthermore, reporter assays showed that mutations in EVI1 altered both AP-1- and TGFb-mediated transcriptional responses. These findings suggest that transcriptional dysregulation by mutant EVI1 could be associated with the development of ATRUS. Consequently, we report missense mutations in MECOM resulting in a Mendelian genetic disorder that provide compelling evidence for the critical role of EVI1 in normal hematopoiesis and in the development of forelimbs and fingers in humans.
2014 - ACUTE MYELOID LEUKEMIA THERAPEUTIC TARGET DECONVOLUTION BY COMPOUND CLUSTERING AND SYNERGISTIC FINGERPRINTING Irene Baccelli1,2,3, Jana Krosl1, Genevieve Boucher1, Laura Simon1, Josee Hebert1, Sebastien Lemieux1, Anne Marinier1, and Guy Sauvageau1 1 University of Montreal, Montreal, Canada; 2IRIC, Montreal, Canada; 3 The Leucegene project, Montreal, Canada Chemotherapeutic agents currently used for the treatment of Acute Myeloid Leukemia (AML) are in most cases unable to eradicate the disease. The low number of therapeutic breakthroughs in this area in the last decades stems in particular from i) the hitherto lack of appropriate in vitro culture methods for patient leukemic stem cells and ii) the scarcity of innovative analytical approaches. In this study, we present a novel two-pronged methodology for the identification of efficient therapies in AML relying on i) compound correlation clustering and ii) synergistic fingerprinting. Using in vitro culture conditions recently uncovered for the maintenance of leukemic stem cell activity, we interrogated genetically diverse AML patient specimens with a small library of off-patent compounds. Hierarchical clustering analysis by icicle representation led to the identification of groups of molecules belonging to one or several different chemotypes inducing similar inhibitory patterns across samples, which we named Compound Correlation Clusters (CCCs). These CCCs likely reveal distinct therapeutic targets in AML. While a minority associated with the presence of specific mutations, the majority identified new therapeutic classes. CCC synergistic fingerprints were surprisingly recurrent across specimens, suggesting that therapeutic targets are synchronous and that only a handful of combinatorial treatments might be sufficient to treat the majority of patients. Our study may represent a revolutionary approach to deconvolute large numbers of compounds into small relevant entities that can be analyzed for synergistic interactions in cell-based assays. We anticipate that it will be a starting point for the development of novel personalized treatment strategies in AML or in other diseases, with a particular interest in patients with poor prognostic.
S45
2016 - SUBCLONAL EVOLUTION OF PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA REVEALED BY GENETIC BARCODING Mirjam Belderbos1,2, Gerald de Haan3, Taco Koster3, Vincent van der Velden4, and Leonid Bystrykh3 1 Dept. of Stem Cell Biology and Ageing, European Research Institute for the Biology of Ageing, The Netherlands; 2Dept. of Pediatrics, University Medical Center Groningen, The Netherlands; 3European Research Institute for the Biology of Ageing, Groningen, The Netherlands; 4Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands Introduction: B-cell precursor acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children. Although most patients obtain complete remission, relapse occurs in w15-20% of patients accounting for high mortality. Genetic heterogeneity and clonal evolution are thought to drive leukemia progression and relapse. The frequency of leukemia-propagating cells (LPC), their genetic diversity and the dynamics of their resulting subclones remain incompletely characterized, which is in part due to the use of different clonal markers. Aim: Here, we aim to characterize the frequency of LPC in pediatric B-ALL in an in vivo model using three different markers: Immunoglobulin heavy chain (IgH) rearrangements, leukemia-associated genetic mutations and DNA barcodes. Methods: Diagnostic bone marrow cells from pediatric patients with high-risk B-ALL (BCR-ABL or delIKZF1) were genetically barcoded using a library of w800 barcodes incorporated into a lentiviral vector, and serially transplanted into immune deficient NOD-SCID-IL2Rg-/- mice. Barcode composition and IgH gene rearrangements were determined by PCR and deep sequencing on blood at several time points after transplantation, and in bone marrow, liver and spleen at sacrifice. Genetic mutations were measured by quantitative realtime PCR. Results: Highly-sensitive IgH analysis identified only 2-3 subclones which remained present in primary, secondary and tertiary xenografts. In contrast, barcode analysis revealed hundreds of LPC clones in primary xenografts. Barcode complexity decreased dramatically upon serial transplantation, to a median of 3 (2-4, p ! 0.001) barcode subclones in tertiary recipients. Interestingly, all tertiary recipients had the same dominant subclone, suggestive of its enhanced fitness. In xenografts of the BCR-ABL positive leukemia, we identified an IKZF-deletion in secondary xenografts, of which the presence increased in tertiary xenografts, indicating ongoing acquisition of genetic mutations that may drive subclonal evolution. Conclusion: Pediatric B-ALL in an individual patient consists of hundreds of LPC capable of propagating disease in murine xenografts. Combining different clonal markers is essential to obtain a comprehensive view on leukemia subclonal evolution. Future research is aimed at identifying the genetic and functional characteristics of dominant subclones, which may benefit the diagnosis, prevention and treatment of relapsed leukemia.
missense mutations in MECOM, encoding EVI1. Subsequent analysis of MECOM in two other individuals with ATRUS revealed two additional missense mutations. These three mutations (Thr756Ala, His751Arg, Arg750Trp) were clustered within the 8th zinc finger motif of the C-terminal zinc finger domain of EVI1. Chromatin immunoprecipitation and quantitative polymerase chain reaction assays of the regions harboring the ETS-like motif that is known as an EVI1 binding site showed a reduction in immunoprecipitated DNA for two EVI1 mutants compared with wild-type EVI1. Furthermore, reporter assays showed that mutations in EVI1 altered both AP-1- and TGFb-mediated transcriptional responses. These findings suggest that transcriptional dysregulation by mutant EVI1 could be associated with the development of ATRUS. Consequently, we report missense mutations in MECOM resulting in a Mendelian genetic disorder that provide compelling evidence for the critical role of EVI1 in normal hematopoiesis and in the development of forelimbs and fingers in humans.
2014 - ACUTE MYELOID LEUKEMIA THERAPEUTIC TARGET DECONVOLUTION BY COMPOUND CLUSTERING AND SYNERGISTIC FINGERPRINTING Irene Baccelli1,2,3, Jana Krosl1, Genevieve Boucher1, Laura Simon1, Josee Hebert1, Sebastien Lemieux1, Anne Marinier1, and Guy Sauvageau1 1 University of Montreal, Montreal, Canada; 2IRIC, Montreal, Canada; 3 The Leucegene project, Montreal, Canada Chemotherapeutic agents currently used for the treatment of Acute Myeloid Leukemia (AML) are in most cases unable to eradicate the disease. The low number of therapeutic breakthroughs in this area in the last decades stems in particular from i) the hitherto lack of appropriate in vitro culture methods for patient leukemic stem cells and ii) the scarcity of innovative analytical approaches. In this study, we present a novel two-pronged methodology for the identification of efficient therapies in AML relying on i) compound correlation clustering and ii) synergistic fingerprinting. Using in vitro culture conditions recently uncovered for the maintenance of leukemic stem cell activity, we interrogated genetically diverse AML patient specimens with a small library of off-patent compounds. Hierarchical clustering analysis by icicle representation led to the identification of groups of molecules belonging to one or several different chemotypes inducing similar inhibitory patterns across samples, which we named Compound Correlation Clusters (CCCs). These CCCs likely reveal distinct therapeutic targets in AML. While a minority associated with the presence of specific mutations, the majority identified new therapeutic classes. CCC synergistic fingerprints were surprisingly recurrent across specimens, suggesting that therapeutic targets are synchronous and that only a handful of combinatorial treatments might be sufficient to treat the majority of patients. Our study may represent a revolutionary approach to deconvolute large numbers of compounds into small relevant entities that can be analyzed for synergistic interactions in cell-based assays. We anticipate that it will be a starting point for the development of novel personalized treatment strategies in AML or in other diseases, with a particular interest in patients with poor prognostic.
S45
2016 - SUBCLONAL EVOLUTION OF PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA REVEALED BY GENETIC BARCODING Mirjam Belderbos1,2, Gerald de Haan3, Taco Koster3, Vincent van der Velden4, and Leonid Bystrykh3 1 Dept. of Stem Cell Biology and Ageing, European Research Institute for the Biology of Ageing, The Netherlands; 2Dept. of Pediatrics, University Medical Center Groningen, The Netherlands; 3European Research Institute for the Biology of Ageing, Groningen, The Netherlands; 4Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands Introduction: B-cell precursor acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children. Although most patients obtain complete remission, relapse occurs in w15-20% of patients accounting for high mortality. Genetic heterogeneity and clonal evolution are thought to drive leukemia progression and relapse. The frequency of leukemia-propagating cells (LPC), their genetic diversity and the dynamics of their resulting subclones remain incompletely characterized, which is in part due to the use of different clonal markers. Aim: Here, we aim to characterize the frequency of LPC in pediatric B-ALL in an in vivo model using three different markers: Immunoglobulin heavy chain (IgH) rearrangements, leukemia-associated genetic mutations and DNA barcodes. Methods: Diagnostic bone marrow cells from pediatric patients with high-risk B-ALL (BCR-ABL or delIKZF1) were genetically barcoded using a library of w800 barcodes incorporated into a lentiviral vector, and serially transplanted into immune deficient NOD-SCID-IL2Rg-/- mice. Barcode composition and IgH gene rearrangements were determined by PCR and deep sequencing on blood at several time points after transplantation, and in bone marrow, liver and spleen at sacrifice. Genetic mutations were measured by quantitative realtime PCR. Results: Highly-sensitive IgH analysis identified only 2-3 subclones which remained present in primary, secondary and tertiary xenografts. In contrast, barcode analysis revealed hundreds of LPC clones in primary xenografts. Barcode complexity decreased dramatically upon serial transplantation, to a median of 3 (2-4, p ! 0.001) barcode subclones in tertiary recipients. Interestingly, all tertiary recipients had the same dominant subclone, suggestive of its enhanced fitness. In xenografts of the BCR-ABL positive leukemia, we identified an IKZF-deletion in secondary xenografts, of which the presence increased in tertiary xenografts, indicating ongoing acquisition of genetic mutations that may drive subclonal evolution. Conclusion: Pediatric B-ALL in an individual patient consists of hundreds of LPC capable of propagating disease in murine xenografts. Combining different clonal markers is essential to obtain a comprehensive view on leukemia subclonal evolution. Future research is aimed at identifying the genetic and functional characteristics of dominant subclones, which may benefit the diagnosis, prevention and treatment of relapsed leukemia.