Figure 2.
YKL-05-099 inhibits the growth of MLL-rearranged leukemia cells by modulating SIK3-mediated regulation of HDAC4. (A) Chemical structure of YKL-05-099. (B) Relative growth of the indicated cells using CellTiter-Glo assays. Normalized relative luminescence units (RLU) are shown after 3 days in culture with DMSO (0.1%) or YKL-5-099 at the indicated concentrations. The mean ± standard error of the mean (SEM; n = 3) and 4-parameter dose-response curves are plotted. (C) Scatterplot of SIK2/SIK3 essentiality CRISPR scores from our prior study22 and YKL-05-099 EC50 in the indicated AML cell lines. The CRISPR scores were calculated in cells cotransduced with SIK2 and SIK3 sgRNAs and cell fitness tracked in competition-based assays. (D) Western blot analysis of MEF2C in the indicated AML cell lines. (E) Flow cytometry analyses of DNA content to infer cell cycle status after 24-hour treatment with 1 µM YKL-05-099 or DMSO. (F) Flow cytometry analyses of side scatter (SSC) and annexin-V staining (a preapoptotic cell marker) after 24-hour treatment with 1 µM YKL-05-099 or DMSO. (G) Relative growth of RN2 cells transduced with empty vector, Sik3, or Sik3T142Q cDNA, after YKL-05-099 treatment. Normalized relative luminescence units (RLU) are shown after 3 days of culture with DMSO (0.1%) or YKL-05-099 at the indicated concentrations. The mean ± SEM (n = 3) and 4-parameter dose-response curves are plotted. (H) Bright-field images of methylcellulose-based colony formation assays of UCB cells, AML cell lines, and PDX models with DMSO or 1 µM YKL-05-099 on day 10 after plating. Scale bar, 500 µm. (I) Quantification of the myeloid colonies shown in panel H. Mean ± standard deviation (n = 3). (J) Western blot analysis in MOLM-14 and MV4-11 cells treated with 1 µM YKL-05-099 or DMSO for 6 hours. (K) Western blot analysis in RN2 cells transduced with empty vector, Sik3, or Sik3T142Q cDNA, following treatment with DMSO (0.1%) or 350 nM YKL-05-099 for 2 hours. (L) Accumulated number of Cas9-expressing RN2 cells transduced with the indicated sgRNAs upon treatment with DMSO (0.1%) or 350 nM YKL-05-099. An average of 3 independent experiments is shown. sgNeg1: negative control.

YKL-05-099 inhibits the growth of MLL-rearranged leukemia cells by modulating SIK3-mediated regulation of HDAC4. (A) Chemical structure of YKL-05-099. (B) Relative growth of the indicated cells using CellTiter-Glo assays. Normalized relative luminescence units (RLU) are shown after 3 days in culture with DMSO (0.1%) or YKL-5-099 at the indicated concentrations. The mean ± standard error of the mean (SEM; n = 3) and 4-parameter dose-response curves are plotted. (C) Scatterplot of SIK2/SIK3 essentiality CRISPR scores from our prior study22  and YKL-05-099 EC50 in the indicated AML cell lines. The CRISPR scores were calculated in cells cotransduced with SIK2 and SIK3 sgRNAs and cell fitness tracked in competition-based assays. (D) Western blot analysis of MEF2C in the indicated AML cell lines. (E) Flow cytometry analyses of DNA content to infer cell cycle status after 24-hour treatment with 1 µM YKL-05-099 or DMSO. (F) Flow cytometry analyses of side scatter (SSC) and annexin-V staining (a preapoptotic cell marker) after 24-hour treatment with 1 µM YKL-05-099 or DMSO. (G) Relative growth of RN2 cells transduced with empty vector, Sik3, or Sik3T142Q cDNA, after YKL-05-099 treatment. Normalized relative luminescence units (RLU) are shown after 3 days of culture with DMSO (0.1%) or YKL-05-099 at the indicated concentrations. The mean ± SEM (n = 3) and 4-parameter dose-response curves are plotted. (H) Bright-field images of methylcellulose-based colony formation assays of UCB cells, AML cell lines, and PDX models with DMSO or 1 µM YKL-05-099 on day 10 after plating. Scale bar, 500 µm. (I) Quantification of the myeloid colonies shown in panel H. Mean ± standard deviation (n = 3). (J) Western blot analysis in MOLM-14 and MV4-11 cells treated with 1 µM YKL-05-099 or DMSO for 6 hours. (K) Western blot analysis in RN2 cells transduced with empty vector, Sik3, or Sik3T142Q cDNA, following treatment with DMSO (0.1%) or 350 nM YKL-05-099 for 2 hours. (L) Accumulated number of Cas9-expressing RN2 cells transduced with the indicated sgRNAs upon treatment with DMSO (0.1%) or 350 nM YKL-05-099. An average of 3 independent experiments is shown. sgNeg1: negative control.

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