Non-Oncogene Addiction to BRD in CLL: from JQ1 Response to Resistence

NON-ONCOGENE ADDICTION TO BRD IN CLL: FROM JQ1 RESPONSE TO RESISTENCE

1_{Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, Orbassano,}

Italy

2_{Division of Internal Medicine – Hematology, San Luigi Gonzaga Hospital, Orbassano, Italy} 3_{Depart. Of Oncology, University of Turin, San Luigi Gonzaga Hospital, Orbassano, Italy}

KEY WORDS: Chronic Lymphocytic Leukaemia; targeted therapy; BRDs; JQ1

CONTEXT: Chronic Lymphocytic Leukemia (CLL) is the most common lymphoproliferative disorder.

Despite significant advances in therapy, CLL remains basically incurable. Current therapy consists in immuno-chemotherapy regimens, but rates of relapse disease are high.

OBJECTIVE: Clinical options are still unsatisfactory, improvement in new therapeutic strategies are

required. We particularly focused on a new drug directly targeting bromodomain proteins.

DESIGN: JQ1 (BRDs inhibitor) efficacy was tested in CLL cell lines. Proliferation, cell cycle and

apoptosis were evaluated respectively with CTG technology and Annexin V-FITC/Propidium PE detection by flow cytometry.

SETTING: Our data support the therapeutic potential of JQ1 inhibitors in CLL and dissect resistance

to targeted therapy, in order to anticipate drug resistance in patients.

RESULTS: To explore the non-oncogene addiction to BRDs transcription factors in CLL cell lines, we

assessed the levels of BRDs expression and we studied a BET bromodomain inhibitors (JQ1, recently found associated to specific BRD4 targeting in other leukemia setngs.). JQ1 induced growth inhibition, G0/G1 phase arrest and apoptosis induction in in MEC-1 and EHEB CLL cell lines.

Subsequently, we examined the mechanisms of JQ1-induced response in CLL cells. JQ1 activity is at least and in part mediated by MYC deregulation (as recently reported) in EHEB cell line but we showed no association between JQ1 sensitivity and MYC expression in MEC-1 cell line, suggesting a more complex control of the transcriptional network by bromodomain factors in CLL.

Cell lines were induced to acquired resistance to BET inhibition. In particular, we established JQ1-resistant cell lines by long-term culture of both MEC-1 and EHEB cells in escalating doses of JQ1. In contrast to parental lines, JQ1-resistant cells proliferated normally, even in high JQ1 doses. We then investigated possible mechanisms of resistance, assessing the expression level of BRDs in sensitive or resistant lines. We found that JQ1-resistance is attributable to up-regulation of BRD2, BRD4 and BRD9, suggesting a broader action of JQ1 in the inhibition, and indirectly, in the regulation of expression of different BRDs in CLLs.

CONCLUSIONS: High efficacy of JQ1 in pre-clinical cellular models of CLL supporting new

therapeutic opportunities also in clinical trials to treat CLL patients. Nevertheless our data showed a compensatory mechanism of resistance mediated by BRDs up-regulation, posing the bases for the investigation of new regulatory circuits involving bromodomain proteins.

This work was supported by fouding of Italian Ministero della Salute, Progetto Giovani Ricercatori, and AIRC-Start-Up Grant#2014-15405