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