A Fully MurinizedBCR-ABL+ Acute Lymphoblastic …...A Fully MurinizedBCR-ABL+ Acute Lymphoblastic...
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A Fully Murinized BCR-ABL+ Acute Lymphoblastic Leukemia ModelSantana Sanchez1, Sean Tracy1 Michael A. Farrar1
1Department of Laboratory Medicine and Pathology, Center for Immunology, Masonic Cancer Center, University of Minnesota
ABSTRACT:Cloning Strategy
ST26 in vitro Proliferation
Human and murine BCR-ABL are equally sensitive to Nilotinib
ST26 cells exhibit a pre-B cell phenotype as assessed by flow cytometry
Future Directions
Patients with acute lymphoblastic leukemia (ALL) harboring the BCR-ABL chromosomal translocation have very poor outcomes, with long-term survival rates of only 30%. BCR-ABL+ ALL has historically lowfrequencies of non-synonymous mutations, which has made it difficultto treat with checkpoint blockade immunotherapy. However, the fusionportion of the BCR-ABL translocation itself forms a neoantigen.Peptides derived from this region have been found to be presented inboth MHCI and MHCII contexts in patients with BCR-ABL+ ALL. Murinemodels of BCR-ABL+ ALL have been utilized to study the effectivenessof heterologous peptide immunization against the fusion region coupledwith checkpoint blockade as a treatment strategy for BCR-ABL+ ALL.These models involve injecting p19Arf-/- pre-B cells that express ahuman BCR-ABL into an immunocompetent mouse. This is followed bytreatment with heterologous peptide immunization coupled withcheckpoint blockade, which significantly enhanced survival (Manlove etal. 2016).The problem with this model is that there are numerous aminoacid discrepancies between the human protein sequence and and themurine sequence. Therefore, the human BCR-ABL fusion proteincontains multiple potentially immunogenic epitopes outside of thefusion region, that could be partially responsible for the anti-leukemiaeffect unlocked by heterologous immunization. We designed a BCR-ABL fusion gene using fully murine genomic DNA. The murine BCR-ABL fusion gene was transduced into primary bone marrow cellsharvested from a mouse lacking the p19Arf-/- gene. After 20 days animmortalized population grew out of culture (ST26 cells). These cellswere characterized by flow cytometry and determined to be of pre-Bcell lineage. In vitro assay showed that the cell line was sensitive totyrosine kinase inhibitors indicating that the proliferation of these cells isdependent on BCR-ABL signaling. Taken together, these data indicatethat ST26 represents a BCR-ABL+ Pre-B cell leukemia cell line that canbe used to validate the heterologous immunization + checkpointblockade immunotherapy strategy for treating BCR-ABL+ B-ALL.
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
C’-F “Pre-B”A-C “Pro-B”
D “Pre-B” 78.6
E-F 18.4
C-D
C-DC’-F “Pre-B”
E-F3.67
D “Pre-B” 95.2A-C “Pro-B”
Cel
l Cou
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Lymphocytes à Singlets à Viable
• Inject ST26 cells intravenously and perform survival analysis. • Identify tissue localization of ST26 cells in mouse. • Characterize ST26 cells to confirm that they display pre-B cell
phenotype in an in vivo context. • Confirm that ST26 cells elicit BAp-specific CD4 T-cells. • Compare T-cell response elicited by ST26 cells head-to-head
with previously established huBCR-ABL+ B-ALL murine models.
Faderl et al. 1998, Blood
Acknowledgements:Farrar Lab
Michael FarrarSean TracyCan Hekim
Hrishi VenkateshRobin Lee
Lucy SjaastadDavid Owen
Lynn Heltemes HarrisGrey Hubbard
p19Arf-/-, huBCR-ABLST26
Cel
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Cel
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t(9;22) Translocation Generates BCR-ABL Fusion Oncoprotein
Red: Aligns with reference sequence (gray). White: Non-aligned regions.
T35 Medical Student Summer Research Program in Infection and ImmunityDaniel L. Mueller
Stephanie Krischuk