Post on 15-Sep-2020
Murinized BCR-ABL+ B-lineage Acute Lymphoblastic Leukemia
Santana SanchezFarrar Lab8/21/19
Background
• B-ALL represents a cancer with very few nonsynonymous mutations suggesting that it is not a good candidate for treatment with checkpoint blockade-based immunotherapy
t(9;22) translocation
BCR-ABLUncontrolled Proliferation
Wikipedia
ABLBCR
Background
• B-ALL represents a cancer with very few nonsynonymous mutations suggesting that it is not a good candidate for treatment with checkpoint blockade-based immunotherapy• Peptides derived from BCR-ABL are antigenic
Background
• B-ALL represents a cancer with very few nonsynonymous mutations suggesting that it is not a good candidate for treatment with checkpoint blockade-based immunotherapy• Peptides derived from BCR-ABL are antigenic• Previous model: Arf-/- Pre-B cells express human BCR-ABL fusion protein
recapitulates BCR-ABL+ B-ALL in an immunocompetent mouse (LM138 cells)
Background
• B-ALL represents a cancer with very few nonsynonymous mutations suggesting that it is not a good candidate for treatment with checkpoint blockade-based immunotherapy
• Peptides derived from BCR-ABL are antigenic
• Previous model: Arf-/- Pre-B cells express human BCR-ABL fusion protein recapitulates BCR-ABL+ B-ALL in an immunocompetent mouse (LM138 cells)
• Manlove et al. 2016 showed that robust immunization with fusion peptide + checkpoint blockade led to significantly improved survival
Background
• B-ALL represents a cancer with very few nonsynonymous mutations suggesting that it is not a good candidate for treatment with checkpoint blockade-based immunotherapy
• Peptides derived from BCR-ABL are antigenic
• Previous model: Arf-/- Pre-B cells express human BCR-ABL fusion protein recapitulates BCR-ABL+ B-ALL in an immunocompetent mouse (LM138 cells)
• Manlove et al. 2016 showed that robust immunization with fusion peptide + checkpoint blockade led to significantly improved survival
• What is the problem?
Background
• B-ALL represents a cancer with very few nonsynonymous mutations suggesting that it is not a good candidate for treatment with checkpoint blockade-based immunotherapy
• Peptides derived from BCR-ABL are antigenic
• Previous model: Arf-/- Pre-B cells express human BCR-ABL fusion protein recapitulates BCR-ABL+ B-ALL in an immunocompetent mouse (LM138 cells)
• Manlove et al. 2016 showed that robust immunization with fusion peptide + checkpoint blockade led to significantly improved survival
• What is the problem?
Background
• B-ALL represents a cancer with very few nonsynonymous mutations suggesting that it is not a good candidate for treatment with checkpoint blockade-based immunotherapy• Peptides derived from BCR-ABL are antigenic• Previous model: Arf-/- Pre-B cells express human BCR-ABL fusion protein
recapitulates BCR-ABL+ B-ALL in an immunocompetent mouse (LM138 cells)• Manlove et al. 2016 showed that robust immunization with fusion peptide
+ checkpoint blockade led to significantly improved survival • Because of discrepancies between human and mouse BCR and ABL protein
sequence there are multiple foreign epitopes on the BCR-ABL fusion protein that are potentially driving the antileukemia immune response seen in Manlove et al.
Goal
• Generate muBCR-ABL+ B-ALL cell line• Proliferate independently in culture• Dependent on BCR-ABL tyrosine kinase activity • Pre-B cell phenotype• Recapitulate B-ALL in vivo
muBCR-ABL
Primary Bone Marrow Cells
Retroviral plasmid
Arf-/-
Cloning Strategy
muBCR-ABL geneblock 1 muBCR-ABL geneblock 2
NotI EcoRI
IRES Thy1.1
NotI EcoRI
Carrier Plasmid
Retrovirus
muBCR-ABL
~20 days
Primary Bone Marrow Cells
muBCR-ABL
Retroviral plasmid
Arf-/-Arf-/-
ST26
muBCR-ABL
~20 days
Characterize by Flow Cytometry
Characterize in vivo
In vitro studies
Primary Bone Marrow Cells
muBCR-ABL
Retroviral plasmid
Arf-/-Arf-/-
ST26
Goal
• Generate muBCR-ABL+ B-ALL cell line• Proliferate independently in culture• Dependent on BCR-ABL tyrosine kinase activity • Pre-B cell phenotype• Recapitulate B-ALL in vivo
ST26 Proliferation
Goal
• Generate muBCR-ABL+ B-ALL cell line• Proliferate independently in culture• Dependent on BCR-ABL tyrosine kinase activity • Pre-B cell phenotype• Recapitulate B-ALL in vivo
ST26 cells are sensitive to Abl kinase inhibitor Nilotinib
muBCR-ABL, ARF-/-(ST26)
huBCR-ABL, ARF-/-(LM138)
Goal
• Generate muBCR-ABL+ B-ALL cell line• Proliferate independently in culture• Dependent on BCR-ABL tyrosine kinase activity • Pre-B cell phenotype• Recapitulate B-ALL in vivo
Goal
• Generate muBCR-ABL+ B-ALL cell line• Proliferate independently in culture• Dependent on BCR-ABL tyrosine kinase activity • Pre-B cell phenotype
• B220+• BP1+• CD19+• IgM-
• Recapitulate B-ALL in vivo
HSC MLP NF B
PreBlate
PreBearlyCLP DJ-
ProBGL-ProB
FoB
AA4.1B220CD43CD24BP-1c-kitIL-7RCD19CD25IgM
Phenotypic Fraction: A B/C C’ D E F
Surf
ace
Prot
eins
Hardy 2004, B cell Protocols
B cell lineage
HSC MLP NF B
PreBlate
PreBearlyCLP DJ-
ProBGL-ProB
FoB
AA4.1B220CD43CD24BP-1c-kitIL-7RCD19CD25IgM
Phenotypic Fraction: A B/C C’ D E F
Surf
ace
Prot
eins
Hardy 2004, B cell Protocols
B cell lineage
WT
BM
ST26
D “Pre-B” 78.6E-F 18.4
D-F “Pre-B”E-F 3.67
D “Pre-B” 95.2
B220
IgM
B220
B220
CD43 IgM
D-F “Pre-B”
B220
CD43
C-D
CD25
BP1
A-C’ “Pro-B”
C-D
CD25
BP1
D-F “Pre-B”
B220
CD43
A-C’ “Pro-B”
Goal
• Generate muBCR-ABL+ B-ALL cell line• Proliferate independently in culture• Dependent on BCR-ABL tyrosine kinase activity • Pre-B cell phenotype
üB220+üBP1+üCD19+ü IgM-
Goal
• Generate muBCR-ABL+ B-ALL cell line• Proliferate independently in culture• Dependent on BCR-ABL tyrosine kinase activity • Pre-B cell phenotype• Recapitulate B-ALL in vivo
Conclusions
üGenerate muBCR-ABL+ B-ALL cell lineüProliferate independently in cultureüDependent on BCR-ABL tyrosine kinase activity üPre-B cell phenotype? Recapitulate B-ALL in vivo
Future Direction
• Survival analysis• Perform flow cytometry analysis on in vivo ST26 cells• Determine how ST26 cells respond to robust immunization +
checkpoint blockade therapy
Acknowledgements
Thank you to the Farrar Lab!Michael FarrarSean TracyCan HekimHrishi VenkateshRobin LeeLucy SjaastadDavid OwenLynn Heltemes HarrisGrey Hubbard
T35 Medical Student Summer Research ProgramDaniel L. Mueller
Stephanie Krischuk