Fig 2: Aberrant CD96 expression in JMML (related to Fig. 6). (Ai) Histograms of CD96 expression on the aberrant Lin-CD34+CD38-CD90+CD45RA+ cells from patient samples (ID1, ID3, ID5, ID6, ID14, and ID15) and Lin-CD34+CD38-CD90+ cells from a normal CB sample. Dashed line represents the CD96 gate. Six biological replicates; representative data shown from one experiment. (Aii) CD96 expression by FACS in CB Lin-CD34+CD38-CD90+ and JMML Lin-CD34+CD38-CD90+CD45RA+ samples showing percentage of cells expressing CD96 (JMML, n = 12; CB, n = 4). Mann-Whitney test; ** P = 0.0049. Error bars represent mean ± SEM. Data shown from two independent experiments. (Aiii) Representative FACS plots of CD96 expression on the aberrant Lin-CD34+CD38-CD90+CD45RA+ cells from patient samples (ID3 and ID5) and CD96 expression of Lin-CD34+CD90+ cells from a normal CB sample. Representative data shown from one experiment. (B) Histogram showing CD96 expression on Lin-CD34+CD90+ cells of a JMML sample (ID19) compared with pediatric BM at diagnosis and 1 mo after transplantation. Dashed line represents the CD96 gate. Patient subsequently relapsed, requiring a second BMT 6 mo after the first BMT. Representative data from one experiment. (C) Representative FACS profile of CD96 expression on mature hematopoietic populations in three JMML patients (ID2, ID5, and ID6). Dashed line represents the CD96 gate. Representative data from one experiment. (D) Representative FACS plots of CD96 expression on Lin-CD34+CD90+ cells in JMML (ID13), chronic phase MPN (CML), and transformed MPN (tMPN AML). Representative data from one experiment. FSCA, forward scatter area.

Fig 3: Conservation of molecular hierarchy of JMML HSPCs and novel therapeutic targets for JMML HSCs. (A) Nearest neighbor analysis of purified HSPC populations from CB and JMML. Numbers represent significant differentially expressed genes <0.1 false discovery rate (FDR). JMML BM, n = 6 different biological replicates, and normal CB control, n = 5 different biological replicates. (B) Heatmap of top differentially expressed genes between JMML HSC, JMML +/+, and JMML GMP. (C–F) Gene expression for CB HSC (black), CB GMP (gray), JMML HSC (blue), JMML CD90+/CD45RA+ (+/+; orange), and JMML GMP (red) for known stem cell genes (C); myeloid genes up-regulated in JMML and CB GMP (D); cell cycle–related genes (E); and expression in JMML HSPCs of known fetal HSC genes (F). (G) GSEA of JMML HSCs versus JMML GMPs with up-regulation of gene sets for HSC markers and CML quiescence in HSCs (top) and up-regulation of gene sets for pediatric cancer and CML proliferation gene sets in GMPs (bottom). NES, normalized enrichment score. (H) Heatmap depicting k-means clustering of top differentially expressed genes between JMML HSCs and CB HSCs with an FDR cutoff <0.1. Cluster size corresponds to the number of genes within each significant cluster. (I) Hallmark GSEA between JMML HSCs and CB HSCs depicting up-regulated gene sets in JMML. Dashed line represents an FDR cutoff of <0.1; numbers next to the bars represent NES. (J) Identification of a JMML LSC–specific gene signature from bulk RNA-seq. (K) Single-cell gene expression of the JMML stem cell genes identified in Fig. 5 J. Clusters are as identified in Fig. 2 B. Expression plotted as mean UMI. (L) Heatmap of top differentially expressed genes between JMML HSC, JMML +/+, and JMML GMP. Data acquired over two independent experiments. TPM, transcripts per million.

Fig 4: Analysis of CD90 transfer by CIMVs-MSCs to recipient HEK293FT cells. (A–G) Laser scanning confocal microscopy using Zeiss LSM 780 (Carl Zeiss, Oberkochen, Germany), (H–I) flow cytometry using BD FACS Aria III (BD Bioscience, San Jose, CA, USA). Green fluorescence—recipient HEK293 FT cell stained with DiO; red fluorescence—parental MSCs or CIMVs-MSCs stained with DiD, blue fluorescence—cells stained with anti-CD90 antibody. (A–C) MSCs stained with DiD and anti-CD90 antibody. (D–G) HEK293FT cells treated with CIMVs-MSCs (CIMVs-MSCs—red spots) and stained with DiO and anti-CD90 antibody.

Fig 5: CD96 expression in JMML HSPCs. (A) CD96 expression by bulk RNA-seq in JMML and CB HSCs, P < 0.05 Benjamini-Hochberg–corrected false discovery rate. Data acquired through bulk RNA-seq. (B) CD96 expression by single-cell RNA-seq (left) on tSNE analysis from CB and JMML (right). (C) Violin plots of single-cell CD96 expression in JMML and CB CD34+ HSPCs. (D) CD96 expression by FACS in CB and JMML Lin-CD34+CD90+ cells showing percentage of Lin-CD34+90+ cells expressing surface CD96 (JMML, n = 19 different biological replicates; CB controls, n = 11 different biological replicates). Data acquired over five independent experiments. Statistical analysis performed by Mann-Whitney test, **** P < 0.0001. Error bars represent mean ± SEM. (E) Presence of Lin-CD34+CD90+CD96+ cells in paired diagnostic and posttransplant samples in patient ID5 detected by flow cytometry. Data acquired over two independent experiments; representative plots from one experiment shown. Dashed line represents the CD96 gate. (F) Dot plot showing correlation between CD96 expression on Lin-CD34+CD90+ cells from JMML patients (age > 6 mo) at diagnosis and fetal hemoglobin (HbF) percentage at the same time point. Pearson's correlation and P value generated by ggplot2 and ggExtra R package (P = 0.0043). JMML, n = 17 different biological replicates. Data acquired over five independent experiments. (G) CD96 expression by FACS in Lin-CD34+CD90+ cells in JMML patient samples (n = 19 different biological replicates), CB controls (n = 11 different biological replicates), and different pediatric and adult myeloid malignancies (MPN: essential thrombocytopenia, n = 3 different biological replicates; myelofibrosis, n = 3 different biological replicates; CML, n = 3 different biological replicates; polycythemia vera, n = 3 different biological replicates; MPN transformed to AML (tMPN AML), n = 5 different biological replicates; and pediatric AML cases, n = 3 different biological replicates. **** P < 0.0001. Error bars represent mean ± SEM. Data acquired over six independent experiments. (Hi) Percent human CD45+ cells in PB from mice transplanted with CD96+ (red line) or CD96- (black dashed line) Lin-CD34+CD38- cells. Data acquired from one independent experiment. Error bars represent mean ± SEM. (Hii) Kaplan-Meier leukemia-free survival curve of mice transplanted with purified JMML Lin-CD34+CD38- CD96+ (n = 1,802 cells per recipient) or JMML Lin-CD34+CD38- CD96- cells (n = 382 cells per recipient). Three animals transplanted per group. Significance by log-rank test, * P = 0.025. TPM, transcripts per million.