Figure 1. Conditional knockout of Kat2a promotes differentiation of MLL-AF9-transformed cells in vitro.

(A) Serial re-plating of colony-forming cell (CFC) assays of MLL-AF9 transformed cells, mean ± SEM, n = 3. (B) Excision efficiency was evaluated by qPCR during re-plating of MLL-AF9 transformed cells, mean ± SEM, n = 2–3, *p<0.01 and **p<0.001. (C) Proportion of Compact-type colonies in MLL-AF9 transformed cells on Kat2a WT or KO background, mean ± SEM, n = 3, *p<0.01 and **p<0.001. (D) Representative photograph of a Compact-type colony. (E) Flow cytometry analysis of the colony in (D). (F) Proportion of Mixed-type colonies in MLL-AF9 transformed cells on Kat2a WT or KO background, mean ± SEM, n = 3, *p<0.01 and **p<0.001. (G) Representative photograph of a Mixed-type colony. (H) Flow cytometry analysis of the colony in (G). Two-tailed t-test was performed in (A), (B), (C) and (F).

Figure 1—figure supplement 1. Loss of Kat2a does not affect normal hematopoiesis.

(A) Diagram of conditional Kat2a floxed (WT) and Kat2a-excised (KO) alleles, including excision detection strategy and diagrams of the transcript and protein generated upon Kat2a locus excision. Kat2a IN (In11, within excised region) and Kat2a OUT primers (Out18, downstream of excised region) used in qPCR analysis of genomic DNA. Amplicons generated by primer pairs Ex1−2/Ex2 (red), Ex2/Ex18(2) (blue) and Ex18 (green) distinguish WT from KO transcript. (B) Excision efficiency quantified by qPCR in mouse BM samples, mean ± SEM, n = 4, **p<0.01. (C) Quantitative RT-(q)PCR analysis of Kat2a transcript levels in BM LMPP and GMP; mean ± SEM, n = 4, **p<0.001. (D) RT-qPCR analysis of red, blue and green amplicons in A for diagnosis of Kat2a WT and KO AML samples, mean ± SEM, n = 4. (E) Flow cytometry analysis of stem and progenitor BM composition in Kat2a WT and KO young mice (6 weeks after pIpC treatment), mean ± SEM, n = 3’ *p<0.05. (F) Colony-forming assays of progenitor populations (left to right: HSC, MPP, GMP and MEP) isolated from Kat2a WT and KO BM 4–6 weeks after excision, mean ± SEM, n = 4–5, *p<0.05. (G) Flow cytometry analysis of stem and progenitor BM composition in Kat2a WT and KO old mice (>4 months after pIpC treatment), mean ± SEM, n > 5. (H) Flow cytometry analysis of donor-derived BM stem and progenitor cells in a long-term hematopoietic reconstitution assay. Irradiated recipients were transplanted with Kat2a WT or KO cells and analyzed 16–20 weeks later; mean ± SEM, n = 4–5. Two-tailed t-test was performed in (B), (C), (E) and (F).

Figure 1—figure supplement 2. Loss of Kat2a promotes differentiation of MLL-AF9 leukemia cells in vitro.

(A) Representative image of compact colonies obtained from lineage-depleted BM cells transduced in vitro with a retrovirus encoding the leukemic MLL-AF9 fusion genes and plated serially through three methylcellulose-based colony-forming assays. (B) Flow cytometry plot of the colonies in (A). (C) Representative image of a mixed colony obtained from lineage-depleted BM cells transduced in vitro with a retrovirus encoding the leukemic MLL-AF9 fusion genes and plated serially through three methylcellulose-based colony-forming assays. (D) Flow cytometry plot of the colonies in (C). (D) CFC assay frequency, mean ± SEM, n = 3, **p<0.001. (E) Proportion of colony types in CFC assays from clonal liquid cultures initiated with Kat2a WT vs KO cells transformed in vitro by MLL-AF9-expressing retroviral particles, mean ± SEM, n = 3, *p<0.01 and **p<0.001. (F) Mean fluorescence intensity of Mac1 in Kat2a WT and KO cells obtained from clonal liquid cultures, mean + SEM, n = 3, ***p<0.0001. Two-tailed t-test was performed in (E) and (F).