Inhibition of bromodomain and extra‐terminal proteins increases sensitivity to venetoclax in chronic lymphocytic leukaemia

Abstract The development of drugs able to target BTK, PI3k‐delta and BCL2 has dramatically improved chronic lymphocytic leukaemia (CLL) therapies. However, drug resistance to these therapies has already been reported due to non‐recurrent changes in oncogenic pathways and genes expression signatures. In this study, we investigated the cooperative role of the BCL2 inhibitor venetoclax and the BRD4 inhibitor JQ1. In particular, we found that JQ1 shows additional activity with venetoclax, in CLL cell lines and in ex vivo isolated primary CD19+ lymphocytes, arguing in favour of combination strategies. Lastly, JQ1 is also effective in venetoclax‐resistant CLL cell lines. Together, our findings indicated that the BET inhibitor JQ1 could be a promising therapy in CLL, both as first‐line therapy in combination with venetoclax and as second‐line therapy, after the emergence of venetoclax‐resistant clones.

It should be noted that in all the trials with these new drugs in the CLL context, idelalisib, ibrutinib and venetoclax are always used as single agents. 9 In the dynamic process of clonal evolution of cancer, the administration of a single agents could simply represent a selective pressure to promote the evolution of sub-clones, thereby selecting those with a resistant behaviour.
It is indeed mandatory to design combinatorial strategies to be administrated upfront the development of resistant clones or alternatively move into a step by step (drug by drug) process.
Venetoclax resistant clones originate from cells where a set of genes are differentially expressed when compared with parental clones. Among these, the major determinants are MCL-1 and BCL-X L. 10,11 This means that, parallel to apoptosis promotion through BCL2 modulation, venetoclax affects the overall gene expression machinery. Some of these genes may favour cells to survive.
In line with these considerations, a perfect drug to associate with venetoclax would be a drug that transiently impairs gene expression during venetoclax administration, preventing the activation of compensatory mechanisms that inhibit or affect the induction of apoptosis.
The bromodomain and extra terminal BET family proteins comprise four members (BRD2, BRD3, BRD4 and the testis-specific BRDT), which contain tandem bromodomains that allow to interact with acetylated lysines of histones and regulate gene transcription of relevant oncogenes, such as MYC, CDK genes, cyclin D1 and BCL2. [12][13][14] Currently, various BET inhibitors have been developed and few of them are under investigation in clinical trials. 15,16 However, while BET inhibitors were consistently described as highly effective drugs in in vitro experiments, few in vivo observations and early clinical trial reports had already posed some concerns regarding the potential development of resistance to BET inhibitors, when administrated as single agents. 16 In the case of AML, for instance, treatment with BET inhibitors promotes resistance through the up-regulation of BET target genes. 17 In this report, we have investigated the mechanisms of venetoclax resistance in CLL. In addition, we have demonstrated the efficacy of BET inhibitors, JQ1, in combination with venetoclax and against venetoclax resistant clone. and with obscured identity, as processed as previously described. 20 Briefly, CLL cells were isolated from peripheral blood samples by density gradient centrifugation accordingly to Ficoll procedure (Sigma-Aldrich). Following centrifugation at 400 g for 20 minutes, CD19+ lymphocytes were isolated accordingly to the Miltenyi Biotec protocol (Miltenyi Biotec, #130-050-301).

| Cells and primary human samples
The project was reviewed and approved by the institutional ethical committee (code #10/2013).

| Gene expression analysis
RNA was extracted from cells using TRIzol as described. 21 1 μg of total RNA was used for reverse transcription with iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's protocol.

The combination index (CI) for drug combination was calculated
with the available software CalcuSyn. CI values < 1.0 indicate a synergistic interaction of the two drugs in the combination.

| Anchorage-independent cell-growth assay
Cells were suspended in 0.45% type VII low-melting agarose in 10% IMDM at a density of 5 × 10 3 cells/well and plated on a layer of 0.9% type VII low-melting agarose in 10% IMDM in 6-well plates then cultured at 37°C with 5% CO 2 . After 2 weeks, colonies were counted, and images were acquired at 5× magnification.

| Treatment with JQ1 inhibits growth and survival in CLL cell lines
We first determined the JQ1 effect on the growth and survival of both

| Combined treatment with JQ1 and venetoclax exerts synergistic lethal activities against CLL cell lines
To define whether pharmacological inhibition of BRD4 synergies with venetoclax regimen, we treated both TP-p53-mutated-MEC1 and TP-p53 wild-type-EHEB cells with a combination of venetoclax plus JQ1, observing a significantly higher reduction in cell viability compared to single treatments (Figure 2A,B). Moreover, due to the ability of JQ1 to modulate BCL2 expression, 22 we observed that the association with venetoclax plus JQ1 favours a profound BCL2 downmodulation ( Figure 3C,D) which may be the cause of such a remarkable apoptosis induction ( Figure 2E,F and Figure S2A). Notably, treatment with JQ1 and venetoclax in combination synergistically induced apoptosis of CLL cell lines, with combination index (CI) <1.0 ( Figure S2B). To recapitulate the mechanism of this synergy, it is worth to note that in some previously published works, it was showed that EGFR and insulin receptor families are direct targets of BRD4 in several cancer models. This strongly suggests that, by blocking BRD4, BET inhibition can regulate the PI3K signal in cancer cells. 23 However, in xenograft models of endometrial cancer, JQ1 significantly increases the expression of PTEN, favouring the block of the PI3K/AKT signalling pathway. 24 Very recently it was demonstrated that BRD4 inhibition induces apoptosis and growth arrest in glioblastoma cells via VEGF/PI3K/AKT regulation. 25 Furthermore, JQ1 is also effective to reduce the levels of p-Erk 1/2 in mouse models of pancreatic cancer and in anaplastic thyroid cancer cells where is often overactivated. Similar results were obtained also in ovarian cancer cells. [26][27][28] Accordingly, Western blot analysis showed that only JQ1 and not venetoclax reduced phosphorylation of ERK1/2 and AKT, potentially explaining the synergy of drugs in impairing CLL survival from a regulatory point of view ( Figure 2G,H).

Altogether, these data indicate that treatment of CLL cells with
BETi enhances the susceptibility to venetoclax.

| Co-treatment with JQ1 and venetoclax affects the viability of ex vivo human CLL cells
To assess whether the combinatorial therapy may affect primary CD19 positive lymphocytes from CLL patients, we treated freshly isolated CD19 positive CLL cells with JQ1, and venetoclax alone or

| JQ1 treatment is effective against venetoclaxresistant CLL cells
Lastly  Figure 4B). Colony formation assay further supports the resistance of MEC-1 cells to venetoclax ( Figure 4C).

ti-tumour effect of BRD inhibition in venetoclax-resistant cells could
depend on an impairment of this protein activity ( Figure 4G). Lastly, by treating primary CD19 cells collected from a patient with venetoclax resistant disease, the apoptotic response is slightly increased ( Figure   S4A,B). Overall, these data suggest that BRD inhibition could be an intriguing second-line therapy regimen in CLL, in the case of emergence of venetoclax resistance, but further investigations are needed.

| D ISCUSS I ON
Dissecting the process of resistance to targeted therapies is impor- Lastly, it should be also considered that therapies with venetoclax, as with ibrutinib and idelalisib, appear to last indefinitely. An intensification schedule may eventually offer the change to limit the duration of the therapy allowing to eradicate the disease rather than controlling it.

ACK N OWLED G EM ENTS
We thank all the member of the Medicine and Molecular Oncology laboratory of the San Luigi Hospital for helps and supports. This