Retention of CD19 intron 2 contributes to CART-19 resistance in leukemias with subclonal frameshift mutations in CD19

Mukta Asnani^#¹, Katharina E Hayer^#^{1

2}, Ammar S Naqvi^{1

2}, Sisi Zheng^{1

3}, Scarlett Y Yang¹, Derek Oldridge^{3

4}, Fadia Ibrahim⁴, Manolis Maragkakis^{4

5}, Matthew R Gazzara⁶, Kathryn L Black^{1

7}, Asen Bagashev¹, Deanne Taylor^{2

8}, Zissimos Mourelatos⁴, Stephan A Grupp^{3

8}, David Barrett^{3

8}, John M Maris^{3

8}, Elena Sotillo^{1

9}, Yoseph Barash⁶, Andrei Thomas-Tikhonenko^{10

11

12}

Affiliations

¹ Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
² DBHi Bioinformatics Group, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
³ Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
⁴ Department of Pathology & Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA.
⁵ Laboratory of Genetics and Genomics, National Institute on Aging, NIH, Baltimore, MD, USA.
⁶ Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA.
⁷ Lonza Biologics, Portsmouth, NH, USA.
⁸ Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA.
⁹ Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA.
¹⁰ Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA. andreit@pennmedicine.upenn.edu.
¹¹ Department of Pathology & Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA. andreit@pennmedicine.upenn.edu.
¹² Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA. andreit@pennmedicine.upenn.edu.

^# Contributed equally.

PMID: 31591467
PMCID: PMC7214268
DOI: 10.1038/s41375-019-0580-z

Retention of CD19 intron 2 contributes to CART-19 resistance in leukemias with subclonal frameshift mutations in CD19

Mukta Asnani et al. Leukemia. 2020 Apr.

. 2020 Apr;34(4):1202-1207.

doi: 10.1038/s41375-019-0580-z. Epub 2019 Oct 7.

Authors

Affiliations

¹ Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
² DBHi Bioinformatics Group, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
³ Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
⁴ Department of Pathology & Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA.
⁵ Laboratory of Genetics and Genomics, National Institute on Aging, NIH, Baltimore, MD, USA.
⁶ Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA.
⁷ Lonza Biologics, Portsmouth, NH, USA.
⁸ Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA.
⁹ Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA.
¹⁰ Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA. andreit@pennmedicine.upenn.edu.
¹¹ Department of Pathology & Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA. andreit@pennmedicine.upenn.edu.
¹² Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA. andreit@pennmedicine.upenn.edu.

^# Contributed equally.

PMID: 31591467
PMCID: PMC7214268
DOI: 10.1038/s41375-019-0580-z

No abstract available

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Conflict of interest statement

SAG, DB, JMM, ES, and ATT have an interest in intellectual property “Discovery of CD19 Spliced Isoforms Resistant to CART-19.” This interest does not meet the definition of a reviewable interest under Children’s Hospital of Philadelphia’s (CHOP’s) conflict of interest policy and is therefore not a financial conflict of interest. Furthermore, this intellectual property is held by both CHOP and the University of Pennsylvania and has not been licensed or otherwise commercialized to date. However, should this technology be commercialized in the future, they would be entitled to shares of royalties earned by CHOP per its patent policy.

Figures

**Fig. 1**
Robust retention of intron 2 limits CD19 protein expression. a Schematic representation of the retroviral construct containing N-terminal VSVg-tagged CD19 coding sequence including the two introns flanking exon 2. U3, R, and U5 form the retroviral long terminal repeats (LTR); SD and SA denote 5′ (“donor”) and 3’ (“acceptor”) splice sites, respectively; MCS denotes the multiple cloning site. b Splicing of the retrovirally encoded CD19 transcript. The CD19 retroviral cassette was transfected into 293T cells, transcribed, spliced, and packaged into viral particles, which were used to infect a CD19-null derivative of the Raji B-cell line. The transduced Raji cells were sorted into CD19-positive and negative populations after gating based on the parental cell line. The splicing pattern of CD19 exon 2 was analyzed by genomic DNA PCR using the VSVg-specific forward primer to prevent amplification of the endogenous CD19 locus. The identity of all PCR products was confirmed by Sanger sequencing. c Long-read RNA-Seq analysis of the endogenous CD19 transcript using the Oxford Nanopore Technology (ONT). The library was prepared from 1 µg of poly(A)-enriched mRNA from REH B-ALL cells using Direct RNA sequencing kit (SQK-RNA002). It was loaded onto a R9.4 flow cell and sequenced on the MinION device FLO-MIN106D for 48 h. The fast5 files were processed using the Guppy algorithm (v3.2.2). Alignment to the human genome (hg19) was achieved using minimap2 (v2.17-r941). The FLAIR package (v1.4) was used to create a collapsed view of the different CD19 isoforms. The structure of the CD19 transcript was visualized using Integrative Genomic Viewer (IGV). The corresponding fastq file has been deposited in GEO under accession number GSE136068. d Profiling of ribosomes on the endogenous CD19 transcript. The whole cell lysate was prepared by incubating 2 × 10⁷ REH cells in lysis buffer supplemented with 100 µg/ml cycloheximide. It was layered onto a 10-50% sucrose density gradient, subjected to centrifugation at 35,000 rpm for 3 h, and fractionated into twenty four 0.5-ml fractions. The top panel shows the ribosomal content measured at 254 nm. The bottom panel shows the relative distribution of specific transcripts across the gradient calculated using the formula 2^{Ct (fraction 1-X)} X 100/sum

**Fig. 2**
Contribution of CD19 intron 2 retention to resistance to CART-19. a MAJIQ output for screening samples CHOP101 and CHOP105R1 compared with relapse samples CHOP101R and CHOP105R2 (top and bottom, respectively). The diagram at the top represents key splicing events. The color-matched violin plots represent the abundance of individual splice isoforms. Double asterisk denote the 0.99 probability of at least 5% difference in percent-spliced-in values (∆psi). b RT-qPCR analysis of CD19 intron 2 retention in CHOP101 and CHOP101R samples using oligonucleotides that span constitutive exon/exon junctions (ex4–5 and ex3–4) and a cassette exon/intron junction (ex2-in2). The RQ values were normalized for CD19 expression levels measured using ex3–4 primers, as described previously by Sotillo et al. c IGV visualization of CD19 transcripts in CHOP101 and CHOP101R samples using as sources untrimmed bam files. Coverage tracks show read coverage for a given gene segment. Junction tracks summarize reads spanning junctions denoted by arches. The red dotted oval denotes the subclonal frameshift insertion. d IGV visualization of CD19 transcripts in 5S and 5R samples using as sources trimmed bam files deposited in the Short Reads Archive as SRR7353764 and SRR7353766. The red dotted oval denotes the subclonal frameshift mutation. Other designations are as in c

See this image and copyright information in PMC

References

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Retention of CD19 intron 2 contributes to CART-19 resistance in leukemias with subclonal frameshift mutations in CD19

Affiliations

Retention of CD19 intron 2 contributes to CART-19 resistance in leukemias with subclonal frameshift mutations in CD19

Authors

Affiliations

Conflict of interest statement

Figures

References

Publication types

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Grants and funding

LinkOut - more resources

Full Text Sources

Medical

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