Abstract 333: BRD4 inhibitor-resistant acute myeloid leukemia cells develop new sensitivities and resistances to small-molecule inhibitors identifiable through high-capacity drug screening
Authors: Romine Kyle A. , Kurtz Stephen , Tyner Jeffrey 
Published: August 2019

Abstract
Acute Myeloid Leukemia (AML) has a poor prognosis for patients who cannot tolerate conventional chemotherapy, which has prompted the development of many small-molecule inhibitors now in clinical trials. Recently, inhibitors targeting bromodomain containing protein 4 (BRD4i), an epigenetic regulator of many proliferative genes, have shown promise in AML based on in vitro and ex vivo activity. Mechanisms through which AML cells develop resistance to epigenetic modifying drugs such as BRD4i has not been thoroughly evaluated and its elucidation will provide insight into the epigenetic states of AML as well as resistance mechanisms that could lead to combination therapies with broader and more durable activity. The aim of this study is to 1) Develop AML cells resistant to 3 BRD4i (JQ1, OTX-015, and CPI-0610) with similar mechanisms-of-action, 2) Screen a panel of small molecules to identify inhibitors that are differentially effective in BRD4i-resistant lines compared to parental, 3) Identify changes in signaling pathways by evaluating the phosphorylation state of proteins in resistant versus parental cells. Results: We developed BRD4i-resistant versions of the OCI-AML2 (DNMT3a) and MOLM13(FLT3-ITD) cell lines by passaging the cells for 3 months under increasing selective pressure of the three inhibitors, reaching a minimum of 5 fold resistance to the BRD4i. We found that these cell lines were pan-resistant to the BRD4i, such that a cell line treated with JQ1 was also more resistant to OTX-015 and CPI-0610. Drug sensitivity changes in BRD4i-resistant OCI-AML2 show a 90-fold increased resistance to the BCL-2 inhibitor, venetoclax. BRD4i-resistant MOLM13 cells also have a 10-fold increased resistance to venetoclax. To evaluate the phosphorylation status of many oncogenic proteins simultaneously, phosphokinase arrays were performed on parental and resistant cells with and without treatment of drug. JQ1-resistant OCI-AML2 cells have significantly reduced phosphorylation of CREB and p38α. Additionally, these cells have reduced sensitivity to the CREB inhibitor 666-15. In contrast, MOLM13 CPI-0610-resistant cells showed a dramatic increase in CREB phosphorylation compared to parental cells, indicating dysregulation of CREB pathway activation state may be important in developing BRD4i resistance. This may also explain resistance to venetoclax, as activated CREB mediates the transcription of BCL-2. Conclusions: Our work demonstrates the potential of AML cell models of BRD4i-resistance to evaluate changes in oncogenic kinase signaling as well as the rapid identification of alternative therapeutic strategies that may have immediate clinical benefit. In addition, we have begun to understand at a mechanistic level how cancer cells evade apoptosis induced by BRD4 inhibition.
Citation Format: Kyle A. Romine, Stephen Kurtz, Jeffrey Tyner. BRD4 inhibitor-resistant acute myeloid leukemia cells develop new sensitivities and resistances to small-molecule inhibitors identifiable through high-capacity drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 333.