Fig 1: CA suppresses growth of JAK2-mutant AML by inhibiting phosphorylation of STAT1 S727.A. Immunoblots after 2 h treatment showing dose-dependent inhibition of STAT1 S727 phosphorylation by CA in SET-2 cells. Ruxolitinib (Rux), a JAK1/2 inhibitor, inhibits tyrosine phosphorylation of STAT1 at 1 µm. Combined treatment with 1 µm ruxolitinib and 50 nM CA. Representative blots shown of at least three biological replicates.B. Competitive growth experiments show that SET-2 and UKE-1 cells overexpressing the phosphomimetic mutant STAT1 S727E (ZsGreen) outcompete cells expressing wild-type STAT1 (mCherry) in the presence of CA (mean ± s.e.m. ratio, n = 3 biological replicates). Representative growth curves shown for experiments repeated at least three times.C. A homozygous CRISPR-Cas9-edited clone expressing STAT1 S727E is resistant to CA compared to a control CRISPR knockout line targeting AAVS1. Solid lines represent calculated non-linear fit (STAT1 S727E(GI50) = 37 nM, sgAAVS1(GI50) = 0.3 nM). Dots represent the mean ± s.e.m. ratio of n = 3 biological replicates.D. CA-induced upregulation of CD61 can be inhibited by STAT1 S727E overexpression. CD61 mRNA transcript levels were measured by ddPCR (mean ± Poisson error, n = 5 biological replicates, experiment performed twice, P = 0.000091, two-tailed t-test).E. STAT1 S727A overexpression causes upregulation of CD41 on the cell surface compared to wild-type STAT1 in SET-2 cells. Representative flow cytometry traces shown and quantification plotted on the right (mean ± s.e.m., n = 4 biological replicates).See also Fig. S2.
Fig 2: Inhibition of CDK8/CDK19 reduces proliferation and promotes differentiation of JAK-STAT activated AML and AMKL.A. JAK2-dependent AML cell lines SET-2 and UKE-1, and AMKL cell line 6133/MPL are highly sensitive to CDK8/19 inhibition by cortistatin A (CA). Ba/F3 pro-B cells expressing JAK2 V617F or MPL W515L are resistant to CA. Doses of CA causing 50% growth inhibition (GI50) calculated from full-dose response curves from at least three separate experiments with three biological replicates per dose.B. Ruxolitinib-persistent cell lines SET-2Per and UKE-1Per retain sensitivity to CA (mean ± s.e.m. of n = 3 biological replicates). Representative growth curves shown for experiments repeated at least three times.C. CA and ruxolitinib show synergy in SET-2 and UKE-1 cells. Cells were treated with CA alone, ruxolitinib alone, and CA and ruxolitinib in 1:1 or 1:10 ratios for 10 days and combination index values were assessed at 50% growth. Each dot represents one full independent experiment in which synergy was assessed.D. CA promotes megakaryocytic differentiation in SET-2 cells after 3 days of treatment. Representative flow cytometry traces shown. Quantification of mean ± s.e.m., n = 3 biological replicates, one of two independent experiments shown.E. CA induces dose-dependent upregulation of the megakaryocyte markers CD41 and CD42 in 6133/MPL cells after 7 days of treatment. Representative flow cytometry traces shown. Quantification of mean ± s.e.m., n = 3 biological replicates, one of two independent experiments shown.F. Competitive growth assays show that SET-2 cells overexpressing CA-resistant CDK8 W105 M or CDK19 W105 M (mCherry) outcompete cells expressing wild-type CDK8 or CDK19 (ZsGreen) in the presence of CA, respectively (mean ± s.e.m. ratio of n = 3 biological replicates). Representative growth curves shown for experiments repeated at least three times.See also Fig. S1. For all figures, *P < 0.05 **P < 0.01 ***P < 0.001 ****P < 0.0001 or ns (not significant).
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