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HEXIM1 induction is mechanistically involved in mediating anti-AML activity of BET protein bromodomain antagonist

Leukemia volume 30, pages 504–508 (2016)Cite this article

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In the present studies, we first knocked down the mRNA and protein expression of HEXIM1 in the cultured AML MOLM13 and patient-derived primary (p) AML BPCs. As compared with the MOLM13 cells transduced with lentivirus-containing, non-targeted short hairpin RNA (shRNA) (MOLM13-NT cells), MOLM13-HKD (HEXIM1 knockdown) cells transduced with the shRNA specific for HEXIM1 exhibited marked attenuation of the mRNA and protein levels of HEXIM1 (Figure 1b). MOLM13-HKD cells also showed (by confocal immunofluorescence) depletion of the nuclear levels of HEXIM1 (Figure 1b). Notably, as compared with the MOLM13-NT, MOLM13-HKD cells showed greater increase in cell numbers when placed in suspension culture over 96 h (P<0.05) (Figure 1d). Next, we determined the effect of the BA JQ1 on MOLM13-NT versus MOLM13-HKD cells. JQ1 treatment induced significantly more HEXIM1 protein levels in MOLM13-NT versus MOLM13-HKD cells (Figure 1c). The levels of HEXIM2 were undetectable and were not induced by JQ1 (Figure 1c). As compared with MOLM13-NT cells, c-Myc expression was higher in the untreated MOLM13-HKD cells, and treatment with JQ1 attenuated c-Myc levels in MOLM13-HKD cells (Figure 1c). JQ1 treatment was less effective in inhibiting the suspension culture growth of MOLM13-HKD versus MOLM13-NT cells (Figure 1d). JQ1 induced less morphologic features of differentiation and less percentage of differentiated MOLM13-HKD cells, as compared with MOLM13-NT cells (Figure 1e). JQ1 also dose-dependently induced more apoptosis of MOLM13-NT versus MOLM13-HKD cells (Figure 1f). We also transduced the HEXIM1 shRNA into freshly procured pAML BPCs expressing FLT3-ITD (pAML-HKD cells). As shown in Figure 1g, compared with the pAML cells transduced with non-targeted shRNA (pAML-NT cells), pAML-HKD cells expressed markedly lower protein levels of HEXIM1 but higher levels of c-Myc, whereas HEXIM2 levels were similar in the two cell types. Notably, treatment with JQ1 induced HEXIM1 in pAML-NT but not in pAML-HKD cells, whereas HEXIM2 levels remained unperturbed (Figure 1g). JQ1 treatment also attenuated c-Myc levels (Figure 1g). Concomitantly, JQ1 treatment induced differentiation in a greater percentage of pAML-NT BPCs, as compared with pAML-HKD cells (Figure 1h). Moreover, treatment with JQ1 dose-dependently induced significantly more apoptosis of pAML-NT, as compared with pAML-HKD BPCs (Figure 1i).

In the nucleus, the regulatory subunit of pTEFb, cyclin T1, binds to CDK9 or to the C-terminal domain of HEXIM1,11 based on which we determined the effect of HEXIM1 knockdown on the binding of cyclin T1 to HEXIM1 and CDK9 in unperturbed and JQ1-treated MOLM13-NT cells. In the unperturbed MOLM13-NT cells, immunoprecipitated cyclin T1 bound to HEXIM1 and CDK9 (Supplementary Figure 1a). Treatment with JQ1 increased the levels and binding of immunoprecipitated cCyclin T1 to HEXIM1, which was not observed in MOLM13-HKD cells (Supplementary Figure 1a). JQ1 treatment also caused increased binding of cyclin T1 to CDK9 in both MOLM13-NT and MOLM13-HKD cells (Supplementary Figure 1a), without causing increase in the levels of cyclin T1 and CDK9 (Supplementary Figure 1b). These results were confirmed by confocal immunofluorescence microscopy, which also demonstrated that the nuclear colocalization of cyclin T1 and HEXIM1 in MOLM13-NT cells was not observed in MOLM13-HKD cells (Supplementary Figure 1c). Moreover, increased nuclear colocalization of cyclin T1 and HEXIM1 in MOLM13-NT cells because of JQ1 treatment was abrogated in MOLM13-HKD cells (Supplementary Figure 1c).

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Acknowledgements

This research is supported in part by the MD Anderson Cancer Center Support Grant (P30 CA016672) and the MD Anderson Cancer Center Leukemia SPORE (CA 100632).

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Authors and Affiliations

  1. Houston Methodist Research Institute, Houston, TX, USA

    S G T Devaraj, B Shah, B Sun & S P Iyer

  2. Baylor College of Medicine, Houston, TX, USA

    W Fiskus

  3. Dana-Farber Cancer Institute, Boston, MA, USA

    J Qi & J E Bradner

  4. University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, USA

    S Sharma

  5. MD Anderson Cancer Center, Houston, TX, USA

    K N Bhalla

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  1. S G T Devaraj
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Correspondence to K N Bhalla.

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Devaraj, S., Fiskus, W., Shah, B. et al. HEXIM1 induction is mechanistically involved in mediating anti-AML activity of BET protein bromodomain antagonist. Leukemia 30, 504–508 (2016). https://doi.org/10.1038/leu.2015.142

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