![The CRISPRKMT2A::AFF1 iALL model recapitulates variation in CD133 expression, and CD133 status correlates with differences in functional and molecular signatures. (A) PROM1 TPM from hCD45+CD19+ cells from bone marrow of NSG mice transplanted with Cas9-only (control, n = 4) and CRISPRKMT2A::AFF1 leukemias (transformed, n = 17) fetal liver CD34+ cells (P = .02). PROM1 high (above median) and low (below median) leukemias are indicated by brackets. (B) Correlation of PROM1 gene expression and CD133 protein expression by flow cytometry for CRISPRKMT2A::AFF1 leukemias (n = 17, R2 = 0.92). (C) CD133 positivity in CD19+ blasts in samples of patients with iALL at diagnosis (n = 11) and relapse (n = 5) compared to CRISPRKMT2A::AFF1 leukemias (n = 17). (D) Survival curves of NSG mice with CD133+ (defined as >20% of blasts CD133+) or CD133– primary CRISPRKMT2A::AFF1 leukemias (n = 4 CD133+, n = 12 CD133–, log-rank P = .0004). (E) Volcano plot of differential gene expression between PROM1 high and low CRISPRKMT2A::AFF1 leukemias. Genes with adjusted P < .05 and log-fold change >0.58 or less than –0.58 are shown in yellow and blue, respectively. Myeloid/lymphoid program associated genes are labelled in red and green, respectively. (F) Gene set enrichment analysis (GSEA) reaching statistical significance (false discovery rate [FDR] 0.05) from fetal entries in MSigDB C8 (cell type signature gene sets18), CRISPRKMT2A::AFF1 PROM1 high vs low. (G) GSEA of a core stem cell gene set,19CRISPRKMT2A::AFF1 PROM1 high vs low (normalized enrichment score = 1.63, adjusted P ≤ .001). (H) GSEA reaching statistical significance (FDR, 0.05) from entries in MSigDB H1 (hallmark gene sets), CRISPRKMT2A::AFF1 CD133+ vs CD133– blasts. (I) GSEA of a composite core stem cell gene set,19CRISPRKMT2A::AFF1 CD133+ vs CD133– blasts (normalized enrichment score = 2.88, adjusted P ≤ .001). (J) Uniform manifold approximation and projection (UMAP) of single-cell transcriptomic data of CD19+ blasts from 4 samples of patients with KMT2A::AFF1, displaying log1p (normalized PROM1 gene expression) (n = 2, chALL; n = 2, iALL, total of 14 927 cells). (K) Dot plot showing PROM1 expression pattern at a single-cell level in the 4 KMT2Ar patient samples in panel J. (L) Top 25 most enriched gene set entries in MSigDB H1 (hallmark gene sets) by GSEA, PROM1+ vs PROM1– blasts from KMT2A::AFF1 primary patient samples analyzed by single-cell (sc) RNA-sequencing in panel J. AKT, protein kinase B; chALL, childhood ALL; IL, interleukin; mTORC1, mammalian target of rapamycin complex 1; mTOR, mammalian target of rapamycin; ns, not significant; NES, normalized enrichment score; padj, P adjusted; PI3K, phosphoinositide 3-kinase; TPM, transcripts per million; TNFA, tumor necrosis factor alpha.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/9/18/10.1182_bloodadvances.2024015185/1/blooda_adv-2024-015185-gr1b.jpeg?Expires=1760648827&Signature=vHye58y85tSzHnQ~boGQnyW5Yi5~iY0dtwcQaB8Ci7X7TWwZPy8bqpRaI3ITAXKKWGrQmK8jSVPrdDZWy0pkjUHaBGD0~iZv46Gp0ZEDt2fOzb6tdFbWstmGWQLmNvIuXFqYfFZbVOL5clUkjuF-LzkPyKnfYGPRUMPSLIOR919nr2FprUBv54Au2XUKDUML5D-6w8Z38ydftIsN0QXNKbjbAXNb~AZGgNSykltDRLD9lgLdysqbvilxtZXArLaz-zVQ7UBVpXUvzY22KSgL5qsx277nS5-oe48e-4wPp2Xc77QtTQtqAjOU8ahKlO3Wxp5-qf4StpQBZm1WNJQOBQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The CRISPRKMT2A::AFF1 iALL model recapitulates variation in CD133 expression, and CD133 status correlates with differences in functional and molecular signatures. (A) PROM1 TPM from hCD45+CD19+ cells from bone marrow of NSG mice transplanted with Cas9-only (control, n = 4) and CRISPRKMT2A::AFF1 leukemias (transformed, n = 17) fetal liver CD34+ cells (P = .02). PROM1 high (above median) and low (below median) leukemias are indicated by brackets. (B) Correlation of PROM1 gene expression and CD133 protein expression by flow cytometry for CRISPRKMT2A::AFF1 leukemias (n = 17, R2 = 0.92). (C) CD133 positivity in CD19+ blasts in samples of patients with iALL at diagnosis (n = 11) and relapse (n = 5) compared to CRISPRKMT2A::AFF1 leukemias (n = 17). (D) Survival curves of NSG mice with CD133+ (defined as >20% of blasts CD133+) or CD133– primary CRISPRKMT2A::AFF1 leukemias (n = 4 CD133+, n = 12 CD133–, log-rank P = .0004). (E) Volcano plot of differential gene expression between PROM1 high and low CRISPRKMT2A::AFF1 leukemias. Genes with adjusted P < .05 and log-fold change >0.58 or less than –0.58 are shown in yellow and blue, respectively. Myeloid/lymphoid program associated genes are labelled in red and green, respectively. (F) Gene set enrichment analysis (GSEA) reaching statistical significance (false discovery rate [FDR] 0.05) from fetal entries in MSigDB C8 (cell type signature gene sets18), CRISPRKMT2A::AFF1 PROM1 high vs low. (G) GSEA of a core stem cell gene set,19 CRISPRKMT2A::AFF1 PROM1 high vs low (normalized enrichment score = 1.63, adjusted P ≤ .001). (H) GSEA reaching statistical significance (FDR, 0.05) from entries in MSigDB H1 (hallmark gene sets), CRISPRKMT2A::AFF1 CD133+ vs CD133– blasts. (I) GSEA of a composite core stem cell gene set,19 CRISPRKMT2A::AFF1 CD133+ vs CD133– blasts (normalized enrichment score = 2.88, adjusted P ≤ .001). (J) Uniform manifold approximation and projection (UMAP) of single-cell transcriptomic data of CD19+ blasts from 4 samples of patients with KMT2A::AFF1, displaying log1p (normalized PROM1 gene expression) (n = 2, chALL; n = 2, iALL, total of 14 927 cells). (K) Dot plot showing PROM1 expression pattern at a single-cell level in the 4 KMT2Ar patient samples in panel J. (L) Top 25 most enriched gene set entries in MSigDB H1 (hallmark gene sets) by GSEA, PROM1+ vs PROM1– blasts from KMT2A::AFF1 primary patient samples analyzed by single-cell (sc) RNA-sequencing in panel J. AKT, protein kinase B; chALL, childhood ALL; IL, interleukin; mTORC1, mammalian target of rapamycin complex 1; mTOR, mammalian target of rapamycin; ns, not significant; NES, normalized enrichment score; padj, P adjusted; PI3K, phosphoinositide 3-kinase; TPM, transcripts per million; TNFA, tumor necrosis factor alpha.
