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Letters to the Editor
gene’s transcription start site (TSS), specifically bound in MLL-PTD cells but not in HSPC and could be suppressed by at least 25% through JQ1 treatment. Then, the genes close by such BRD4 binding sites and presenting the same pattern in their expression were classified as posi- tive response to treatment, whereas the genes that pres- ent the opposite trend in their expression were classified as negative response to treatment (Figure 2C). As result, we identified genes whose expression was positively or negatively regulated by changes in BRD4 binding specific in the MLL-PTD AML sample and could be reversed by JQ1 treatment (Figure 2D). We identified 92 genes which were significantly upregulated in MLL-PTD AML cells through BRD4 binding and which were downregulated by BRD4 inhibition (Online Supplementary Table S1). As an example, Figure 2E shows the expression of ADAMDEC1 which is increased in MLL-PTD AML cells and correlates with binding of BRD4. On the other hand, 38 genes were downregulated by BRD4 in the MLL-PTD AML cells and its inhibition led to a re-expression (Online Supplementary Table S1). We also performed ingenuity pathway analysis (IPA) from the ChIP-RNA integration and identified pathways that are affected by BRD4-medi- ated transcriptional changes (Online Supplementary Table S2). These data suggest that the distinct gene expression profile of MLL-PTD positive AML, is at least partly driven by the transcription factor BRD4, similar to AML cells which harbor a t(v;11q23). In addition, we showed that this aberrant expression can be restored after JQ1 treat- ment. Because we compared only cells from one patient (treated vs. untreated) with HSPC from three pooled CB samples, next we wanted to validate the direct binding of BRD4 to two genes that were upregulated in MLL-PTD AML cells, i.e., ADAMDEC1 and SLAMF8 (Figure 3A). These genes were also shown to have a func- tional role in MLL-PTD cells since knockdown of either of these genes resulted in decreased leukemic cell growth (Online Supplementary Figure S1E). Using a chromatin immunoprecipitation followed by quantitative real-time polymerase chain reaction (qRT-PCR), we show that BRD4 binds to ADAMDEC1 and SLAMF8, and this bind- ing can be repressed by JQ1 treatment. Moreover, both genes are upregulated in MLL-PTD AML patients cells compared to CB cells (Figure 3B). For both genes, JQ1 treatment led to decreased expression. Similar results were found when MLL-PTD was knocked down (Online Supplementary Figure S1F and G). We also found similar results in our in vivo mouse model. Mice treated with JQ1 had lower expression of several potential oncogenes, including Adamdec1 and Slamf8 (Online Supplementary Figure S1H to J). Finally, we showed that JQ1 treatment of MLL-PTD cells also results in the decreased expression of BCL2, CDK6, and MYC, well-established downstream targets of BRD4 (Figure 3C).1,2 We found that knocking down the MLL-PTD fusion gene using a short hairpin RNA (shRNA) also resulted in downregulation of these proteins regulated by BRD4, similar to treatment with JQ1 (Figure 3D). Previously, it has been shown that fusion proteins from t(v;11q23) can initiate aberrant gene expression profiles by recruiting BRD4.1 Our data suggest that in MLL-PTD cells utilize a similar mechanism and could account for the distinct gene expression profile.
Taken together, our data shows that targeting BRD4 with the small molecule JQ1 reduces cell proliferation of MLL-PTD cells in vitro and induces apoptosis. In line, we found that JQ1 treatment decreased leukemic burden in vivo and improved survival in vivo. We show for the first time to our knowledge, that aberrant BRD4 binding in MLL-PTD cells results in a distinct deregulation of genes.
By integrating the RNA-seq and ChIP-seq analysis, we identified targets relevant to the MLL-PTD subgroup of AML patients and validated in additional samples from MLL-PTD+ AML primary patient blasts and our unique AML mouse model. This novel group of genes might be associated with leukemogenesis of MLL-PTD+ CN-AML and also with the poor prognosis of this subgroup. Importantly, we were able to reverse the aberrant gene expression patterns by treatment with JQ1. Therefore, targeting BRD4 might be an effective and promising treatment option for patients harboring a MLL-PTD to improve their outcome.
Marius Bill,1,2* Chinmayee Goda,1* Felice Pepe,1 Hatice Gulcin Ozer,3 Betina McNeil,1 Xiaoli Zhang,3 Malith Karunasiri,1 Rohan Kulkarni,1 Sonu Kalyan,1 Dimitrios Papaioannou,1,4 Gregory Ferenchak,1 Ramiro Garzon,1,4 James E. Bradner,5 Guido Marcucci,6 Michael A. Caligiuri,6
and Adrienne M. Dorrance1,4
*MB and CG contributed equallly as co-first authors.
1The Ohio State University, Comprehensive Cancer Center, Columbus, OH, USA; 2Medizinische Klinik und Poliklinik I, Universitätsklinikum Carl Gustav Carus Dresden, Dresden, Germany; 3The Ohio State University, Department of Biomedical Informatics, Columbus, OH, USA; 4Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA; 5Dana-Faber Cancer Institute, Boston, MA, USA and 6City of Hope Comprehensive Cancer Center, Duarte, CA, USA
Correspondence: ADRIENNE DORRANCE - adrienne.dorrance@osumc.edu
doi:10.3324/haematol.2020.271627 Received: September 8, 2020. Accepted: April 30, 2021. Pre-published: May 13, 2021.
Disclosures: JEB is a shareholder and executive of Novartis AG and provided JQ1 for the studies. All other authors declare no conflicts of interest.
Contributions: AMD designed the study; MB, CG, FP, BM, MK, RK, DP and GF performed the experiments; MB, CG, FP, BM, MK, RK, SK, DP, GF, RG, JB, GM, MAC and AMD contributed to the data interpretation; MB, CG, FP and AMD wrote the manuscript; HGO and XZ performed bioinformatics and statistical analyses.
All authors reviewed the manuscript.
Acknowledgments: the authors would like to thank the patients who consented to participate and the families who supported them;
to Donna Bucci, Christopher Manring and the Leukemia Tissue Bank at The Ohio State University Comprehensive Cancer Center, Columbus, OH, for sample processing and storage services.
References
1. Yue Zhang, Aili Chen, Xiao-Mei Yan and Gang Huang. Disordered epigenetic regulation in MLL-related leukemia. Int J Hematol. 2012;96(4):428-437.
2. Winters AC, Bernt KM. MLL-rearranged leukemias-an update on sci- ence and clinical approaches. Front Pediatr. 2017;5:4.
3. Ballabio E, Milne TA. Molecular and epigenetic mechanisms of MLL in human leukemogenesis. Cancers (Basel). 2012;4(3):904-944.
4. Mrózek K, Marcucci G, Paschka P, Whitman SP, Bloomfield CD. Clinical relevance of mutations and gene-expression changes in adult acute myeloid leukemia with normal cytogenetics: are we ready for a prognostically prioritized molecular classification? Blood. 2007; 109(2):431-448.
5. Whitman SP, Liu S, Vukosavljevic T, et al. The MLL partial tandem duplication: evidence for recessive gain-of-function in acute myeloid leukemia identifies a novel patient subgroup for molecular-targeted therapy. Blood. 2005;106(1):345-352.
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