Activity of ipilimumab in bone marrow-involved AML has been modest compared to extramedullary AML relapse or solid tumors like melanoma. Single cell transcriptomics (scRNA-seq) could provide deeper understanding of these differences, yet leukemia and immune cell phenotypes defined by somatic mutations or differential isoform expression often cannot be resolved with native short-read scRNA-seq data due to insufficient read coverage. To overcome this limitation, we devised a workflow ( nanoranger) that leverages improved accuracy of long-read sequencing (Oxford Nanopore, ONT) and enables targeted read-out of molecular features including single-nucleotide variants, gene fusions, isoforms, or T cell receptor (TCR) sequences from intermediate scRNA-seq cDNA libraries. To determine the performance of nanoranger, we undertook a series of assessments. First, we established its genotyping accuracy based on the ability to recover highly polymorphic TCR sequences. TCRs obtained with Illumina versus ONT from the same VD(J) amplicon library were highly concordant ( r>0.8) in the number of reads and cells per CDR3 (5,653 [ONT] vs. 5,767 [Illumina]). Second, we tested isoform profiling by targeted sequencing of PTPRC which encodes the T cell differentiation markers CD45RA (exon 4 present) and CD45RO (exon 4 absent). Not only did targeted ONT sequencing increase the single cell coverage of PTPRC from 10 to >2,600 cells, but PTPRC exon 4 expression also correlated with CD45RA surface marker expression by CITE-seq ( r=0.45). Third, we benchmarked somatic mutation detection of a homozygous TP53 R248Gmutation in the AML cell line Kasumi-1. Our ONT-based approach increased genotyped cells from 0% (via native scRNA-seq) to 22% ( nanoranger); in >98.8% of these cells, TP53 R248G was detectable. Finally, we searched for optimal nanoranger targets by assessing whole-transcriptome coverage of 5‘ single cell cDNA and found these to be loci at the 5‘ end of highly expressed transcripts shorter than 4 kB. Altogether, nanoranger reliably recovers molecular features from scRNA-seq libraries with genotyping efficiency dictated by inherent characteristics of the underlying cDNA. To better understand determinants of response and resistance of CTLA-4 blockade in AML, we applied nanoranger to bone marrow samples collected in the context of a phase I trial investigating combined decitabine and ipilimumab (NCT02890329). We genotyped 11 genes recurrently mutated in AML across 15,258 single cell profiles from 13 samples (5 responders at screening and best response, 3 non-responders at screening). At screening, somatic mutations were detectable in myeloid progenitor, monocytic and dendritic cells but also in erythroid and megakaryocytic progenitor populations, consistent with detected copy number changes. Further, we clarified AML subclones with integrated analysis of somatic mutations and chromosomal aberrations. In AML1019, we found two separate clones defined by presence or absence of RUNX1 R320* and amp(8p) or amp(21p). In AML8007, we tracked differential therapeutic responses. At screening, 3 somatic mutations ( DNMT3A V296M, TP53 C176S, TP53 R282W) and 3 chromosomal aberrations ( amp(1p), del(3p) and del(5q)) were detected. At remission, somatic mutations and del(5q) persisted, but amp(1q) and del(3p) became undetectable except in LMPP-like cells. In all 5 responders, somatic mutations remained detectable in all cellular compartments, revealing that the cytoreductive effect of decitabine/ipilimumab was not accompanied by leukemic clone eradication. The ability of nanoranger to perform isoform profiling enabled us to resolve immune phenotypes. Following CTLA-4 blockade, the PTPRC isoform CD45RA was downregulated in CD4 + and CD8 + T cells, consistent with induction of T cell differentiation by ipilimumab. As CTLA-4 exists in two isoforms, we tracked alternative splicing of exon 3 and observed lower expression of the membranous isoform in T cells from AML bone marrow compared to circulating or tumor-infiltrating lymphocytes in melanoma, pointing to context-specific isoform expression of CTLA-4. In sum, genotyping with nanoranger substantially augmented scRNA-seq analyses of ipilimumab-based therapy in bone marrow AML. This exemplifies the great potential that long-read sequencing-based single cell studies hold to better define response and resistance mechanisms of novel immunotherapies.
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Nov 13, 2024
Ryo Nakayama + 6
Open Access