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CXCR4 and MYC dual targeting in DLBCL
Table 2. Sensitivity of diffuse large B-cell lymphoma cell lines to CXCR4 and BET bromodomain inhibition.
CXCR4 expression (MFI-R)
36 25 41 1 3 52 91 1
4 1 1 46 1
IQS-01.01RS cytotoxic effect (100 μM)
32% 37% 42% 36% 35% 9% 38% 36%
48% 40% 53% 43% 63%
CPI203 cytotoxic effect (0.5 μM)
Combination index
Subtype
GCB
ABC
Cell line
OCI-LY8 SUDHL-4 SUDHL-6 SUDHL-16 WSU-DLCL2 Toledo SUDHL-8 NUDHL-1
OCI-LY3 OCI-LY10 SUDHL-2 U2932 HBL-1
38% 1.1 59% 0.6 47% 0.4 86% 0.5 55% 0.4 43% 0.5 14% 1.1 60% 0.8
75% 0.7 40% 0.8 95% 0.8 31% 0.9 39% 0.6
ABC: activated B-cell; GCB: germinal center B-cell; MFI-R: median fluorescence intensity ratio.
nist. We then analyzed the antitumor activity of this highly active stereoisomer, together with IQS-01.01 (racemic mixture) in six representative DLBCL cell lines of both GCB and ABC subtypes. As shown in Figure 1D, IQS-01.01RS was significantly more active than the racemic mixture at all the doses tested, with the greatest effect being observed at the 100 mM dose (mean cytotox- icity: 44%; range, 42-49%). This effect was mainly attrib- uted to proliferation blockade, as IQS-01.01RS-treated cultures showed low (< 5%) levels of apoptosis (data not shown). In agreement with this, migration experiments using a CXCL12 gradient further demonstrated that IQS- 01.01RS was a more potent inhibitor of cell chemotaxis than was the racemic mixture, with a 2-fold improve- ment of cell migration blockade (Figure 1E). Based on these results, we proceeded to a deeper study of the stereoisomer of choice, IQS-01-01RS.
A predictive docking model showed that this stereoiso- mer localized into the extracellular CXCL12 binding domain of CXCR4, close to the position occupied by AMD3100, and simultaneously interacted with an inner, transmembrane domain of the receptor. This unique fea- ture potentially provides IQS-01.01RS with the ability to inhibit CXCR4 in the presence of the ligand, and to inter- fere with the catalytic activity of the receptor (Figure 2A). Supporting this model, using the representative cell line SUDHL-6 we observed that IQS-01.01RS and AMD3100 had similar CXCR4 occupancy activity (Figure 2B), and had similar anti-migratory properties against a CXCL12 gradient in vitro, both in ABC- and GCB-DLBCL cell lines (Figure 2C). However, IQS-01.01RS triggered superior mobilization of DLBCL cells into the circulating blood of the animals (Figure 2D) and was able to block the prolif- eration of a panel of 13 GCB/ABC-DLBCL cell lines in a time-dependent manner, contrasting with the modest antitumor activity of AMD3100 (40% versus 12% mean cell growth inhibition at 48 h) (Figure 2E and Table 2). In addition, in a set of five DLBCL primary cultures, a dose- dependent induction of apoptosis was observed upon exposure to IQS-01.01RS (Figure 2F), contrasting with the reported inability of AMD3100 to induce cell death.36 Accordingly, the analysis of CXCR4 downstream signal- ing showed that the capacity of IQS-01.01RS to inhibit basal and CXCL12-induced phosphorylation of ERK1/2,
AKT and the AKT downstream kinase GSK3-β, was superior to that of AMD3100 (Figure 2G). Of special interest, IQS-01.01RS, rather than AMD3100, allowed strong downregulation of the MYC proto-oncogene in ABC- and GCB-DLBCL cells (Figure 2H). This down- stream target of CXCR4 with a well-established role in the progression of DLBCL,37 has been reported to depend on AKT phosphorylation level for its stabilization, medi- ated by a GSK3-β-dependent phosphorylation of its Thr58 residue and consequent proteosomal degrada- tion.38,39 Supporting the hypothesis that the downregula- tion of MYC may be consequent to inhibition of the CXCR4-AKT axis in IQS-01.01RS-treated cells, the expression of both p-AKT and MYC was almost com- pletely abrogated within a few minutes after the addition of IQS-01.01RS (Figure 2H) and this phenomenon could be maintained for at least 3 h (data not shown). Collectively, these results indicate that the new CXCR4 inhibitor IQS-01.01RS has a unique structure that enables its binding to two distinct domains of the receptor, con- ferring improved mobilizing properties in vivo, as well as superior antitumor activity in vitro.
IQS-01.01RS cooperates with BET bromodomain inhibition in vitro and in vivo
Based on our observation that IQS-01.01RS had the capacity to downregulate MYC, we further investigated in our 13 DLBCL cell lines whether the compound could cooperate with the BET bromodomain antagonist CPI20332 to block cell proliferation. The combination of each agent induced up to 78% cytoxicity at the optimal doses of 100 mM IQS-01.01RS and 500 nM CPI203, with a mean CI of 0.67, indicative of a synergistic drug interac- tion in both ABC- and GCB-DLBCL cells (Figure 3A and Table 2). As expected, a dramatic reduction in MYC pro- tein levels was observed after exposure to the combina- tion (Figure 3B). We confirmed by quantitative poly- merase chain reaction analysis and cycloheximide protein stability assays that this phenomenon was consequent to a simultaneous CPI203-evoked transcriptional repression of MYC gene and an IQS-01.01RS-mediated destabiliza- tion of MYC protein (Figure 3C,D). Confirming that this effect was not due to a BRD4 inhibitor-mediated dimin- ished production of CXCR4, as previously described in T
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