A Fully MurinizedBCR-ABL+ Acute Lymphoblastic …...A Fully MurinizedBCR-ABL+ Acute Lymphoblastic...
1
A Fully Murinized BCR-ABL+ Acute Lymphoblastic Leukemia Model Santana Sanchez 1 , Sean Tracy 1 Michael A. Farrar 1 1 Department 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 low frequencies of non-synonymous mutations, which has made it difficult to treat with checkpoint blockade immunotherapy. However, the fusion portion of the BCR-ABL translocation itself forms a neoantigen. Peptides derived from this region have been found to be presented in both MHCI and MHCII contexts in patients with BCR-ABL+ ALL. Murine models of BCR-ABL+ ALL have been utilized to study the effectiveness of heterologous peptide immunization against the fusion region coupled with checkpoint blockade as a treatment strategy for BCR-ABL+ ALL. These models involve injecting p19 Arf-/- pre-B cells that express a human BCR-ABL into an immunocompetent mouse. This is followed by treatment with heterologous peptide immunization coupled with checkpoint blockade, which significantly enhanced survival (Manlove et al. 2016).The problem with this model is that there are numerous amino acid discrepancies between the human protein sequence and and the murine sequence. Therefore, the human BCR-ABL fusion protein contains multiple potentially immunogenic epitopes outside of the fusion region, that could be partially responsible for the anti-leukemia effect 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 cells harvested from a mouse lacking the p19 Arf-/- gene. After 20 days an immortalized population grew out of culture (ST26 cells). These cells were characterized by flow cytometry and determined to be of pre-B cell lineage. In vitro assay showed that the cell line was sensitive to tyrosine kinase inhibitors indicating that the proliferation of these cells is dependent on BCR-ABL signaling. Taken together, these data indicate that ST26 represents a BCR-ABL+ Pre-B cell leukemia cell line that can be used to validate the heterologous immunization + checkpoint blockade immunotherapy strategy for treating BCR-ABL+ B-ALL. HSC MLP NF B PreB late PreB early CLP DJ- ProB GL- ProB Fo B AA4.1 B220 CD43 CD24 BP-1 c-kit IL-7R CD19 CD25 IgM Phenotypic Fraction: A B/C C’ D E F Surface Proteins 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-D C’-F “Pre-B” E-F 3.67 D “Pre-B” 95.2 A-C “Pro-B” Cell Count 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 Farrar Sean Tracy Can Hekim Hrishi Venkatesh Robin Lee Lucy Sjaastad David Owen Lynn Heltemes Harris Grey Hubbard p19 Arf-/- , huBCR-ABL ST26 Cell Count Cell Count 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 Immunity Daniel L. Mueller Stephanie Krischuk
Transcript of A Fully MurinizedBCR-ABL+ Acute Lymphoblastic …...A Fully MurinizedBCR-ABL+ Acute Lymphoblastic...
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
nt
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
l Cou
nt
Cel
l Cou
nt
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
