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. 2016 Jul 27:7:12320.
doi: 10.1038/ncomms12320.

CD19 CAR immune pressure induces B-precursor acute lymphoblastic leukaemia lineage switch exposing inherent leukaemic plasticity

Elad Jacoby  1 , Sang M Nguyen  1 , Thomas J Fountaine  1 , Kathryn Welp  1 , Berkley Gryder  2 , Haiying Qin  1 , Yinmeng Yang  1 , Christopher D Chien  1 , Alix E Seif  3 , Haiyan Lei  1 , Young K Song  2 , Javed Khan  2 , Daniel W Lee  1 , Crystal L Mackall  1 , Rebecca A Gardner  4 , Michael C Jensen  4 , Jack F Shern  1 , Terry J Fry  1
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"VSports app下载" CD19 CAR immune pressure induces B-precursor acute lymphoblastic leukaemia lineage switch exposing inherent leukaemic plasticity

"VSports app下载" Elad Jacoby et al. Nat Commun. .

Abstract

Adoptive immunotherapy using chimeric antigen receptor (CAR) expressing T cells targeting the CD19 B lineage receptor has demonstrated marked success in relapsed pre-B-cell acute lymphoblastic leukaemia (ALL). Persisting CAR-T cells generate sustained pressure against CD19 that may drive unique mechanisms of resistance. Pre-B ALL originates from a committed pre-B cell or an earlier progenitor, with potential to reprogram into other hematopoietic lineages. Here we report changes in lineage markers including myeloid conversion in patients following CD19 CAR therapy. Using murine ALL models we study the long-term effects of CD19 CAR-T cells and demonstrate partial or complete lineage switch as a consistent mechanism of CAR resistance depending on the underlying genetic oncogenic driver. Deletion of Pax5 or Ebf1 recapitulates lineage reprogramming occurring during CD19 CAR pressure. Our findings establish lineage switch as a mechanism of CAR resistance exposing inherent plasticity in genetic subtypes of pre-B-cell ALL. VSports手机版.

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"VSports app下载" Figures

Figure 1
Figure 1. Phenotypic alterations in clinical ALL samples following CD19 CAR.
(a,c,e,g) Fever curves, CRP values and clinical response to CD19 CAR treatment. (b,d,f,h) Bar graph demonstrating percent of cells expressing cell surface markers by flow cytometry in the bone marrow, gated on leukaemic blasts, with representative flow cytometry histograms of CD19 and CD11b on the right (grey, control; blue, pre-CAR; red, post CAR). (a,b) Pre-CAR sample and day +30 post-CAR sample from a patient who did not experience CRS following CD19 CAR. (c,d) Pre-CAR and +180 days post-CAR samples from a patient with normal karyotype multiple relapsed ALL, who had a severe CRS followed by an MRD-negative complete response with CD19 CAR, with no CAR+ cells persisting beyond 60 days. (e,f) Pre-CAR and day +30 post-CAR samples from a patient with normal karyotype ALL who experienced a mild CRS and CAR expansion, but had persistent disease. (g,h) Pre-CAR and post-CAR samples from an infant with MLL-rearranged ALL, treated with CD19-41BB-zeta CAR, who relapsed with myeloid blasts.
Figure 2
Figure 2. Distinct phenotypic and genomic alterations in pre-B cell induced by CD19 CAR pressure in vivo.
(a) Schematic design of in vivo murine experiments: mice were injected with leukaemia, followed by lymphodepletion (5 Gy radiation or 4 mg cyclophosphamide) and adoptive T-cell therapy (CAR/Mock T cells) or chemotherapy (4 mg per mouse cyclophosphamide on day 4 followed by 2.5 mg ARAC on days 5 and 10). (b) Survival curve of mice treated with chemotherapy (black, n=5), mock T cells (blue, n=9) or CD19 CAR T cells (red, n=13). Samples marked with φ had no E2a:PBX transgene on PCR and were not further analysed. (c) PCR for E2a:PBX transgene of Eμ-RET cells (negative control), E2A:PBX parental cell line, and splenocytes harvested from leukaemic mice treated with CD19 CAR. CD19 gene (exon2–intron2) was used as control for DNA quantity. (d) Primary and post-CAR relapse leukaemia was analysed by multicolour flow cytometry. Heatmap representing percent of positive cells for multiple lineage markers for each sample. Unsupervised hierarchical clustering is shown above. Mouse IDs as in Supplementary Table 1 are reported below. (e) Quantitative RT PCR for CD19, PAX5 and EBF1 mRNA in primary E2a:PBX leukaemia or mock-treated relapse (n=7, grey), post-CAR CD19− B-ALL relapses (n=2, red) and post-CAR mixed/myeloid relapses (n=7, blue). Data represented as fold change in gene expression over GAPDH, error bars represent s.e.m. *P<0.05, **P<0.01, ***P<0.001 (f) Heatmap demonstrating changes in gene expression of selected hematopoietic transcripts measure by RNA sequencing. Unsupervised clustering is shown above. (g) Principal component analysis of RNA sequencing data from primary and post-CAR samples. Red circle indicates clustering of primary and early-post-CAR samples.
Figure 3
Figure 3. Lineage switch phenotype is not detectable in parental leukaemia and is stable when CAR pressure is removed.
(a) Primary E2a:PBX ALL was single-cell cloned by limiting dilution, expanded and analysed by flow cytometry. Heatmap of cell surface marker expression evaluated by flow cytometry. (b) CD45.2+ B220+ leukaemic blasts from sample 59–61 obtained at day 58 following CAR demonstrating intermediate B/myeloid phenotype expressing for CD22, CD11b and Gr1, but negative for CD19. (c–e) In vivo passage of post-CAR CD19− relapsed leukaemia, with E2a:PBX control, in absence of additional CAR treatment. (c) B/Myeloid biphenotypic sample 25–2 passaged twice off pressure, with re-emergence of a CD19+ population in 16/21 mice in 3rd passage (E2a:PBX control n=16, 2nd n=10, 3rd n=12). (d) Lineage switch samples 24–6 (n=5 per group) and (e) A001 (E2a:PBX control n=3, 2nd n=3, 3rd n=4) passaged twice. Respective survival curves for 1 of 2 experiments shown on the right.
Figure 4
Figure 4. Epigenomic signature demonstrates switch of lineage-specific active chromatin.
ChIP-Seq of H3K27Ac was performed in original disease (E2a:PBX) and four post-CD19-CAR E2a:PBX relapses: a lymphoid CD19-B-ALL (30–4), two mixed B/myeloid samples (5,961 and 252) and a myeloid sample (24–6). (a) ChIP-seq tracks of H3K27ac signal at promoter and enhancer regions for Cd19, Pax5, Ebf1, Sfpi1 (encoding PU.1), Cebpa and Thap2 for the four samples (lymphoid marked in red, myeloid in blue). (b) Transcription factor motif analysis of shared and lineage-specific enhancers was performed, with a more significant P value calculated for more abundant motifs within H3K27Ac-bound chromatin. The Pax5, E2a and Ebf1 motifs were found in regions lost on lineage switch (red box), and absent from newly activated chromatin in samples 59–61, 25–2 and 24–6 (blue box). (c) mRNA expression in fragments per kilobase of transcript per million mapped reads (FPKM) of the transcription factors with enriched motifs (shown in Fig. 4b), grouped according to clustering in Fig. 2d and Supplementary Fig. 5. Error bars represent s.d. *P<0.05.
Figure 5
Figure 5. Knockout of Pax5 or Ebf1 in E2a:PBX pre-B-cell ALL results in a phenotype mirroring relapse under CAR pressure.
(a) Representative structure of Cd19, Pax5 and Ebf1, exons marked in tabs. Guide-RNA design for each knockout shown, with the CAS9 PAM site underlined, and cleavage site marked with a red arrowhead. (b) Flow cytometry plots for murine leukaemia cell lines E2a:PBX (upper panel) and Eμ-RET 309 (bottom panel) following CRISPR/CAS9 knockout of CD19 (green), PAX5 (red), EBF1 (blue) or mock (grey shadow).
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