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SETDB1 expression suppresses MLL-fusion driven AML
ABC
DE
Figure 3. H3K9 methyltransferase inhibitor UNC0638 enhances hematopoietic stem and progenitor cell colony formation capacity. (A-B) Lin- mouse bone marrow was isolated and treated in culture for 4 days with the indicated dose of UNC0638. (A) Cells pretreated with UNC0638 were harvested, lysed in SDS loading buffer, and run on SDS-PAGE. Shown is a Western blot probed for the indicated antibodies. Below the H3K9me2/3 Western blots are numbers indicating the band densities normalized to the Western blot probing for total H3 and normalized to vehicle control. (C) Cells pretreated with UNC0638 were plated in methylcellulose and colonies were counted after 7 days, n=4. Colony numbers are shown relative to the vehicle/non-silencing control for each replicate, bar graphs represent the mean and error bars represent the standard deviation of these normalized values. (C) INT stained representative colonies from (B). (D) Isolated human CD34+ cells were treated for 4 days with the indicated doses of UNC0638 and plated in methylcellulose. Colonies were counted after 14 days, n=2. Colony numbers are shown relative to the vehicle/ non-silencing control for each replicate, bargraphs represent the mean and error bars represent the standard deviation of these normalized values. (E) INT stained representative colonies from (D). Statistics: significance was determined by generalized linear modeling followed by ANOVA where each treated group was paired to the vehicle treatment from the same biological replicate. Main effect is reported if there are no significant interactions (B/D) (See statistical analysis in Online Supplementary Materials and Methods); n: biological replicates; *: P<0.05: INT: 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride; HSPC: hematopoietic stem and progenitor cell.
tent with differentiation (Figure 2E). Genes associated with hematopoietic differentiation, including Id2, Cd80, Nab2, and Itgam have significantly increased expression upon overexpression of SETDB1 (Figure 2F). Additionally, over- expression of SETDB1 in MLL-AF9 cells leads to increased apoptosis (Figure 2G and Online Supplementary Figure S2G). We next examined whether SETDB1 expression is associ- ated with reduced chemoresistance. MA9+SETDB1 do not exhibit reduced apoptosis after treatment with Daunorubicin at their IC50 (Figure 2H and Online Supplementary Figure S2H-I), suggesting SETDB1 expres- sion is not associated with increased Daunorubicin sensi- tivity. Overexpression of another H3K9 methyltransferase, human G9A, also reduces colony formation and prolifera- tion of MLL-AF9 cells (Online Supplementary Figure S3A-C) and induces morphological changes consistent with differ- entiation (Online Supplementary Figure S3D). These data
demonstrate that expression of multiple H3K9 methyl- transferases reduces AML cell proliferation and colony forming potential and induces AML differentiation.
SETDB1 expression delays MLL-AF9 mediated AML
To examine the effects of SETDB1 in vivo, we transplant- ed primary mouse MLL-AF9 AML cells retrovirally trans- duced with or without SETDB1 into sublethally irradiated syngeneic recipient mice and monitored survival. Consistent with AML patient data, overexpression of SETDB1 significantly delays MLL-AF9 mediated leukemo- genesis in vivo (Figure 2I). All moribund mice from both the control MLL-AF9 group and MLL-AF9+SETDB1 group exhibited splenomegaly, leukemic infiltration in the liver (Figure 2J and Online Supplementary Figure S3E) and similar MLL-AF9 expression levels (Online Supplementary Figure S3F). We measured expression of exogenous SETDB1 in
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