![CD133 expression marks KMT2A::AFF1 blasts with higher proliferative potential. (A) Proliferation of CD133+ and CD133–CRISPRKMT2A::AFF1 blasts in MS5 coculture. Data shown as fold change in absolute blast count of CD133+ cells compared to CD133– cells at each time point (n = 4 biological replicates, mean values with error bars = standard error of the mean [SEM]). (B) Representative flow plots from cell cycle analysis of CD133– (left) and CD133+ (right) CRISPRKMT2A::AFF1 blasts on day 7 of coculture. (C) Quantification of cell cycle status of CD133– and CD133+CRISPRKMT2A::AFF1 blasts on day 7 of coculture (n = 4 biological replicates, data shown as mean ± SEM). (D) Percentage of CD133– and CD133+CRISPRKMT2A::AFF1 blasts in sub-G1 phase on day 7 of coculture (n = 4 biological replicates, data shown as mean, error bars = SEM). (E) Survival curves of NSG immunodeficient mice transplanted with CD133+ or CD133–CRISPRKMT2A::AFF1 leukemia cells (4 biological replicates, n = 12 CD133+ animals, n = 11 CD133– animals, log-rank P = .0033). (F) Efficiency of PROM1 KO in CRISPRKMT2A::AFF1 blasts assessed by CD133 surface protein expression by flow cytometry, 7 days postelectroporation (error bars = standard deviation). (G) Left: representative flow plots from cell cycle analysis of PROM1 KO and control CRISPRKMT2A::AFF1 blasts 72 hours after electroporation. Right: percentage of PROM1 KO and control CRISPRKMT2A::AFF1 blasts in sub-G1 phase 72 hours after electroporation (n = 4 biological replicates, data shown as mean, error bars = SEM). (H) Proliferation in MS5 coculture of PROM1 KO and control CRISPRKMT2A::AFF1 leukemia cells. Y-axis values expressed as fold change in absolute blast count of PROM1 KO cells compared to control CRISPRKMT2A::AFF1 cells at each time point (n = 4 biological replicates, error bars = SEM). (I) Top row: survival curves of immunodeficient NSG mice transplanted with PROM1 KO and control CRISPRKMT2A::AFF1 blasts for 2 CD133+ leukemias (n = 3 Cas9 controls and n = 3 PROM1 KO animals for each leukemia; P = .06 and P = .02, respectively). Bottom row: CD133 and CD34 status of blast cells recovered from bone marrow of animals at cull.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/9/18/10.1182_bloodadvances.2024015185/1/blooda_adv-2024-015185-gr2b.jpeg?Expires=1760648829&Signature=S-5jR0fZ2WTOMTWiH0OKNSN2p4LFdiglnqhpVblawtrGdifwsBSnKntAnY11gQddr3LDTgEGAMNEzdQzuvV2GtiVinjDpqctp5lcTJtk7sK77BtuImLh9RAHIUUU~xyKWWKOU9vyzZtupdLJb6RsD6ZDFyhGF4ZvDRdff4x-iJAHJuSIy9S6gX9S5NWK60FBhQKscSqP7N4WKSdEqOcH7yCqxEdTWslQOLt9wl-HI6NISFuDKJ9kLegjXSxQC5F1FI6HgBwpkeLrBnxMtdI0W4FyrRJEo7ol4cAjexHTbQ-WpfyaY4XzYHT-LJdYnvB6zcPzffAZadPQ22R4bSW4FQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CD133 expression marks KMT2A::AFF1 blasts with higher proliferative potential. (A) Proliferation of CD133+ and CD133–CRISPRKMT2A::AFF1 blasts in MS5 coculture. Data shown as fold change in absolute blast count of CD133+ cells compared to CD133– cells at each time point (n = 4 biological replicates, mean values with error bars = standard error of the mean [SEM]). (B) Representative flow plots from cell cycle analysis of CD133– (left) and CD133+ (right) CRISPRKMT2A::AFF1 blasts on day 7 of coculture. (C) Quantification of cell cycle status of CD133– and CD133+CRISPRKMT2A::AFF1 blasts on day 7 of coculture (n = 4 biological replicates, data shown as mean ± SEM). (D) Percentage of CD133– and CD133+CRISPRKMT2A::AFF1 blasts in sub-G1 phase on day 7 of coculture (n = 4 biological replicates, data shown as mean, error bars = SEM). (E) Survival curves of NSG immunodeficient mice transplanted with CD133+ or CD133–CRISPRKMT2A::AFF1 leukemia cells (4 biological replicates, n = 12 CD133+ animals, n = 11 CD133– animals, log-rank P = .0033). (F) Efficiency of PROM1 KO in CRISPRKMT2A::AFF1 blasts assessed by CD133 surface protein expression by flow cytometry, 7 days postelectroporation (error bars = standard deviation). (G) Left: representative flow plots from cell cycle analysis of PROM1 KO and control CRISPRKMT2A::AFF1 blasts 72 hours after electroporation. Right: percentage of PROM1 KO and control CRISPRKMT2A::AFF1 blasts in sub-G1 phase 72 hours after electroporation (n = 4 biological replicates, data shown as mean, error bars = SEM). (H) Proliferation in MS5 coculture of PROM1 KO and control CRISPRKMT2A::AFF1 leukemia cells. Y-axis values expressed as fold change in absolute blast count of PROM1 KO cells compared to control CRISPRKMT2A::AFF1 cells at each time point (n = 4 biological replicates, error bars = SEM). (I) Top row: survival curves of immunodeficient NSG mice transplanted with PROM1 KO and control CRISPRKMT2A::AFF1 blasts for 2 CD133+ leukemias (n = 3 Cas9 controls and n = 3 PROM1 KO animals for each leukemia; P = .06 and P = .02, respectively). Bottom row: CD133 and CD34 status of blast cells recovered from bone marrow of animals at cull.
