CUDC‐907 blocks multiple pro‐survival signals and abrogates microenvironment protection in CLL

Abstract CUDC‐907, a dual PI3K/HDAC inhibitor, has been proposed to have therapeutic potential in hematopoietic malignancies. However, the molecular mechanisms of its effects in chronic lymphocytic leukaemia (CLL) remain elusive. We show that CLL cells are sensitive to CUDC‐907, even under conditions similar to the protective microenvironment of proliferation centres. CUDC‐907 inhibited PI3K/AKT and HDAC activity, as expected, but also suppressed RAF/MEK/ERK and STAT3 signalling and reduced the expression of anti‐apoptotic BCL‐2 family proteins BCL‐2, BCL‐xL, and MCL‐1. Moreover, CUDC‐907 downregulated cytokines BAFF and APRIL and their receptors BAFFR, TACI, and BCMA, thus blocking BAFF‐induced NF‐κB signalling. T cell chemokines CCL3/4/17/22 and phosphorylation of CXCR4 were also reduced by CUDC‐907. These data indicated that CUDC‐907 abrogates different protective signals and suggested that it might sensitize CLL cells to other drugs. Indeed, combinations of low concentrations of CUDC‐907 with inhibitors of BCL2, BTK, or the NF‐κB pathway showed a potent synergistic effect. Our data indicate that, apart from its known functions, CUDC‐907 blocks multiple pro‐survival pathways to overcome microenvironment protection in CLL cells. This provides a rationale to evaluate the clinical relevance of CUDC‐907 in combination therapies with other targeted inhibitors.

homing. 10 Moreover, in this protective niche, bone marrow stromal cells, and NLCs constitutively secrete the chemokines CXCL12 (also known as stroma-cell derived factor, SDF-1) and CXCL13. 11 These activate the surface membrane receptor CXCR4 (sCXCR4) in CLL cells, inducing chemotaxis and migration. 12,13 Multiple signalling pathways, including PI3K/AKT, RAF/MEK/ERK, STAT3, and NF-κB, are constitutively activated in CLL cells. [14][15][16][17][18][19] Given the pivotal role of PI3K/AKT in cancer initiation, growth, proliferation, and survival, an increasing number of small molecular inhibitors that target this pathway have been developed. For example, a PI3Kδ specific inhibitor (CAL-101/idelalisib) has shown promising results in the treatment of patients with relapsed/refractory CLL. 20,21 However, the efficacy of PI3K inhibitors, like most targeted drugs, is limited by concurrent activation of other pro-survival and growth-related pathways that lead to drug resistance. 22 A potential strategy to overcome these limitations is combining PI3K inhibition with drugs that target other pathways, in order to achieve synergistic anti-tumour activity.
HDAC inhibitors can regulate both histone and non-histone protein substrates through epigenetic or non-epigenetic modification, thus affecting multiple signalling networks. 23,24 Several pre-clinical studies of HDAC inhibitors, alone or in combination with other anticancer drugs, have shown promising results for the treatment of hematologic malignancies. 25 For example, simultaneous inhibition of HDAC and PI3K/mTOR signalling with panobinostat and rapamycin has been shown to synergistically inhibit tumour cell growth and induce apoptosis in diffuse large B cell lymphoma (DLBCL) 26 as well as in solid tumour cells. 27 CUDC-907 is a dual inhibitor targeting class I PI3Ks as well as class I and II HDACs, and has been reported to induce apoptosis in various cancer cells in vitro. 28 Recent clinical trials showed the safety, tolerability, and a promising activity of CUDC-907 in patients with relapsed or refractory lymphoma or multiple myeloma, 29 or DLBCL, particularly in MYC mutant malignancies. 30 In this study, we investigated the efficacy of CUDC-907 in CLL cells and found that it effectively disrupted multiple pro-survival signalling pathways to overcome the microenvironment protection and induce apoptosis. This suggest a new mecha- Sigma-Aldrich, Poole, UK) prior to centrifugation at 400 g for 30 minutes. The mononuclear cell layer was removed from the interphase, washed and resuspended in RPMI-1640 medium (Life Technologies, Paisley, UK) supplemented with 10% fetal bovine serum (Lonza, Slough, UK), L-Glutamax (2 mmol/L), penicillin (50 U/mL) and streptomycin (50 mg/mL). The isolated mononuclear cells had a CLL cell purity of >90% in all cases, as determined by flow cytometry.

| Cells, reagents and inhibitors
Chronic lymphocytic leukaemia cells were cultured in RPMI-1640 medium containing soluble 10 ng/mL interleukin (IL)-4 and CD40 ligand (CD40L or CD154) to mimic the microenvironment of proliferation centres 3,4 as previously described. 17 Cells were incubated for 24 hours in these conditions before applying any treatments. Human CLL cell line MEC-1 was cultured as previous described. 17   determined by flow cytometry using a FACS Canto II cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Alternatively, apoptosis was measured by Annexin V staining, as previously described. 17

| Western blotting
Total protein was extracted from cells lysates using RIPA lysis buffer and loading buffer as previous described. 17 Table S1. Relative gene expression was calculated on the basis of the threshold cycle (C t ) values and normalization of internal control expression using the 2 −ΔΔCt method. 32 The internal control housekeeping gene used in this study was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Experiments were performed in triplicate and repeated three times.

| CUDC-907 induces CLL cell death
To uncover potential new therapies for B cell malignancies, we screened the MEC-1 CLL cell line 33 for its response to different clinically relevant targeted inhibitors, including entospletinib (SYK inhibitor), ibrutinib (BTK inhibitor), 34,35 PLX-4720 (BRAF specific inhibitor), CAL-101/Idelalisib (PI3Kδ inhibitor), 20,21 ABT-199 (BCL2 inhibitor), 36 IMD-0354 (NF-κB inhibitor), 37 and CUDC-907 (PI3K/HDAC inhibitor). 28 As shown in Figure 1A and B, CUDC-907 was more effective than all the other inhibitors tested at inducing cell death in the MEC-1 cell line, with an EC50 at nanomolar concentrations. We confirmed these results in CLL patient cells cultured with CD154 and IL-4, a combination of factors that mimics some of the main features of the proliferation centre microenvironment. 38,39 In these conditions, CUDC-907 was shown to be one of the most efficient at reducing cell viability and induce cell death as well, which also happened at nanomolar concentrations and in a dose-dependent manner ( Figure 1C and D). Of note, the sensitivity of CLL cells to BCL-2 inhibitors shown in these figures is consistent with previous reports. 40 These data suggest that CLL cells are highly sensitive to CUDC-907 at concentrations that are within the expected therapeutic levels that can be achieved in vivo, which range from 27 to 70 ng/mL. 29,30 3.2 | CUDC-907-induced CLL cell death is characterized by the inhibition of multiple pro-survival signals We next investigated the mechanisms that could be involved in the effects of CUDC-907 on CLL cell viability. We first tested whether it had any impact on the activation of signalling pathways essential for CLL cell proliferation and survival. As shown in Figure 2A and  8,9 These proteins belong to the TNF superfamily and are important for canonical NF-κB pathway activation. 45 As shown in Figure 2C and Figure S3A, CUDC-907 significantly decreased the expression of all these proteins. We next evaluated the effects of CUDC-907 after BAFF stimulation, in order to assess its impact on the level of NF-κB signalling. As expected, BAFF ligand stimulation induced NF-κB (p65), IκBα, and phospho-IκBα expression, suggesting NF-κB activation ( Figure 2D and Figure S3B). However, CUDC-907 decreased the expression of NF-κB (p65), NF-κB (RelB), IκBα, and phospho-IκBα in a dose-dependent manner. These results together suggest that CUDC-907 inhibits the NF-κB signalling pathway, which may reduce the protection of CLL cells in proliferation centres and thus contribute to the induction of apoptosis.
The CXCR4-CXCL12 axis is critical for the interaction between CLL cells and stromal cells. [10][11][12] We found that CUDC-907 reduced  Figure 3C). This was also evident after stimulation with CXCL12, a ligand of CXCR4 that promotes sCXCR4 receptor internalization. 12 In CLL patient cells, the overexpression of sCXCR4 correlates with hyper-phosphorylated Ser339 residues. 49 We observed that CUDC-907 importantly decreased the phosphorylation level of CXCR4 at Ser339 ( Figure 3D). Total CXCR4 levels were also reduced by CUDC-907 at higher concentrations. Since the binding of CXCL12 to CXCR4 activates different downstream signalling pathways, including RAF/MEK/ERK, 50 we next measured the effect of CUDC-907 on CXCL12-mediated ERK signalling. We found that, while CXCL12 increased ERK phosphorylation, CUDC-907 reduced the levels of P-ERK in both stimulated and unstimulated cells ( Figure 3E).
Together, these data suggest that CUDC-907 impairs CXCL12-CXCR4 signals by decreasing sCXCR4 expression and phosphorylation levels, and thus blocks downstream signalling pathways.

| CUDC-907 enhances CLL cell sensitivity to other targeted inhibitors
Our results so far showed that CUDC-907 is able to suppress different pro-survival signalling pathways often activated in the microenvironment of proliferative centres, while effectively inducing apoptosis through different pathways. This lead to the hypothesis that CUDC-907 could sensitize CLL cells to other drugs by interfering with their protective environment. Thus, we investigated the combination of CUDC-907 with inhibitors that target other signalling pathways, such as those used in Figure 1. We found that the combination of CUDC-907 with IMD-0354, ibrutinib, or ABT-199 increased MEC-1 cell death ( Figure 4A), to the point of being synergistic in most of the concentrations tested (Table S2). The enhancing effect of CUDC-907 on IMD-0354, ibrutinib, and ABT-199 was also observed in CLL patient cells cultured in conditions that mimic the microenvironment of proliferation centres ( Figure 4B). These combinations were also calculated to be synergistic (Table S2). Together, these results suggest that CUDC-907 inhibits many signals involved in the resistance of CLL cells to treatment, which may help sensitize malignant B cells to other targeted treatments. This provides a rationale to further study the possibility of using CUDC-907 as an adjuvant therapy in CLL.

| DISCUSSION
There are many precision medicines currently being assessed clinically to treat B cell malignancies. 22 However, CLL still presents F I G U R E 2 CUDC-907 inhibits pro-survival signals in CLL cells. A, Western blots showing protein expression in lysates of MEC-1 cells treated with CUDC-907 at various concentrations or DMSO as a control (0) for 12 h. AKT, p-AKT (Ser473), p-p70S6K (Thr389), Ac-H3K9, ERK, p-ERK (Thr202/Tyr204), p-MEK (Ser217/221), STAT3, p-STAT3 (Tyr705), MCL-1, BCL-2, BCL-xL, and PARP were detected with specific antibodies. β-actin was used as a loading control. B, Representative Western blot showing PARP, caspase-9, and caspase-8 cleavage in CLL primary cells from two patients. Cells were cultured as in Figure 1C and treated with different concentrations of CUDC-907 for 12 h. C, mRNA expression levels of BAFF, APRIL, BAFFR, TCIA, and BCMA, as measured by qRT-PCR, in CLL patient cells cultured in the presence of 10 ng/mL IL-4 and CD154 for 24 h then treated with CUDC-907 for 12 h. The expression of target genes was normalized to an internal control, GAPDH. Experiments were performed in triplicates and repeated at least 3 times. Data were expressed as relative to control (no inhibitor). D, Representative Western blot of lysates of CLL patient cells stimulated with BAFF (100 ng/mL) for 24 h. NF-κB(p65), NF-κB(RelB), IκBα, p-IκBα, and β-actin were detected using specific antibodies. The graph shows quantitation the Western blot bands normalized to β-actin and expressed as relative to control (0 h) treatment therapeutic challenges, mostly due to the fact that malignant cells are resistant to many drugs when they reside in the bone marrow and lymphoid node microenvironments. It has been confirmed that these protective niches are critical for CLL cells escaping chemotherapy mediated apoptosis. 4 It has also been shown that they are essential for CLL cell proliferation, survival, expansion, and clonal evolution. 7 Thus, blocking the crosstalk of CLL cells with the microenvironment has been proposed to be a key strategy to induce apoptosis. In this study, we reported that CUDC-907, a dual PI3K and HDAC inhibitor, has a strong impact on the microenvironment of malignant B cells and is highly effective in killing CLL cells.
The simultaneous inhibition of HDAC and PI3K signalling has been previously shown to promote the inhibition on tumour cell growth and induction of apoptosis in different cancers, including hematopoietic malignancies. 26,27 Indeed, CUDC-907 has recently been shown to be safe and effective in patients with relapsed/refractory DLBCL. 30 In hematopoietic cells, PI3K signalling activation is normally mediated by the p110δ isoform of PI3K, and it has been suggested that its inhibition has therapeutic value for CLL patients. 21,51,52 CAL-101 (idelalisib), a specific inhibitor of PI3Kδ, has shown a strong effect in the treatment of CLL cells in vitro and in vivo. 20,21 Recently, an inhibitor targeting both isoforms p110δ and p110γ (IPI-145, duvelisib) showed even greater effects than idelalisib in the induction of apoptosis, inhibition of chemokine production, and migratory abilities of CLL cells. 53 Thus, it is still debated whether isoform-specific inhibition, instead of total PI3K inhibition, may be the optimal therapeutic strategy in lymphoid malignancies. In this study, CLL cells showed higher sensitivity to CUDC-907 than idelalisib. This supports the hypothesis that pan-PI3K inhibition may be more effective than blocking specific isoforms.
Our results show that malignant B cells are very sensitive to CUDC-907, which induces cell death by engaging the intrinsic and extrinsic pathways of apoptosis. This is consistent with the fact that HDAC inhibition has previously been reported to induce cell death by various molecular mechanisms, activating both the extrinsic and intrinsic pathways. 23 Apart from its direct apoptotic effects, our results demonstrate that CUDC-907 may also have an impact on survival pathways normally activated in CLL cells. These provide essential proliferation signals for malignant B cells. 14-19 CUDC-907 not only blocked the PI3K/AKT pathway but also simultaneously targeted the RAF/MEK/ERK, STAT3, and NF-κB pathways. Moreover, it also inhibited the expression of anti-apoptotic BCL-2 family proteins such as BCL-2, BCL-xL, and MCL-1. 39,42,43,54 Of note, the protective effects of AKT in CLL cells are largely dependent on the activation of the anti-apoptotic protein MCL-1. 55 The specific mechanisms in stimulated CLL. NF-κB activity normally correlates with chemoresistance and poorer clinical outcome. 14-16 CUDC-907 can inhibit NF-κB signalling and thus reduce the protection that it exerts on CLL cells, which will decrease cell resistance and promote spontaneous or drug-mediated apoptosis. Also, CUDC-907 decreased the mRNA expression and secretion of CCL3 and 4, which are secreted by CLL cells in response to BCR signalling activation or culture with NLS cells and attract T cells to the microenvironment. [46][47][48] CXCR4 is highly expressed on the surface of peripheral blood circulating CLL cells, and is important for CLL cell survival, migration, and interaction with the protective microenvironment. 10,12,56,57 CXCL12 ligands produced by stromal cells induce CLL cell chemotaxis and migration through activation of CXCR4. [11][12][13] In this study, we found that CUDC-907 not only reduced CXCL12-induced smCXCR4 expression but also inhibited CXCR4 phosphorylation at Ser339. This could result in a rapid re-distribution of CLL cells from spleens and lymph nodes into the circulation and prevent re-entry into the microenvironment. 49 This suggests a mechanism to further induce apoptosis in vivo, where CUDC-907 may purge CLL cells from the protective lymph node and bone marrow niches.
Based on all these data, we propose that the pleiotropic effects of CUDC-907 may be critical on its potent impact on CLL cell survival. Moreover, this reduction in pro-survival pathways could help to sensitize CLL cells to other drugs by blocking protective signals. This hypothesis was supported by the fact that we observed a synergistic effect of CUDC-907 with different concentrations of NF-κB inhibitors (IMD-0354), BTK inhibitor (ibrutinib), and BCL2 inhibitors (ABT-199). Recently, we also reported that CUDC-907 can be combined with MEK inhibitors to overcome the resistance of CLL cells to these drugs. 58 The potential combinations of CUDC-907 treatments could therefore be numerous. Importantly, the concentrations of CUDC-907 needed to achieve the synergistic effects described here are in the nanomolar range, which have clinical relevance and would help reduce any off target effects of the drug. We conclude that the ability of CUDC-907 to be combined at very low concentrations with other specific inhibitors to enhance their antitumour activity makes it an interesting candidate for clinical trials in B cell malignancies. Further in vivo experiments will be needed to evaluate this possibility.