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extracted and weighed. Figure 4A shows that CPI203 and IQS-01.01RS as single agents induced 27% and 4% reduc- tions in tumor growth, respectively, while the combina- tion of both drugs induced a 38% decrease in tumor bur- den. Accordingly, reduced luciferase activity as well as a significant (38%) decrease in tumor weight was detected in mice given combination treatment compared to the values in the group administered the vehicle (Figure 4B,C). Histological analysis of the corresponding tumors confirmed a greater reduction of the mitotic index togeth- er with an accumulation of apoptotic cells by the combi- nation therapy, as assessed by phospho-histone H3 and activated-caspase-3 staining (Figure 4D). In agreement with the in vitro results, greater reductions in MYC and phospho-Akt were observed in animals treated with the combination of drugs (Figure 5D). Collectively, these results suggest that the combination of IQS-01.01RS with the BET inhibitor CPI203 enhances the antitumor proper- ties of each single agent through the blockade of CXCR4 signaling, followed by the abrogation of MYC expression and the induction of apoptosis.
Discussion
The chemokine receptor CXCR4 has a prominent role in homing and retention of tumor cells in their microen- vironment, and in the promotion of drug resistance.42 It has been previously reported that CXCR4/CXCL12 expression by tumor cells confers an adverse prognosis to DLBCL patients.12,43 In agreement with this, we observed that CXCL12 expression in tumor biopsies correlated with bone marrow involvement at diagnosis, as well as highly vascularized tumors. These observations support the hypothesis that the CXCL12-CXCR4 axis may play a significant role in neo-angiogenesis in the DLBCL microenvironment. Accordingly, various reports have highlighted a correlation between CXCR4 expression and high serum levels of VEGF in different cancer sub- types.19,21,44 CXCL12 is also one of the genes included in the DLBCL pro-angiogenic and unfavorable “stromal-2 signature”,4 while activation of the CXCL12-CXCR4 axis is related to tumor angiogenesis and recruitment of endothelial progenitor cells to tumors in myelodysplastic syndrome, glioma and pancreatic cancer.20,45,46
The CXCL12-CXCR4 axis may, therefore, represent a new therapeutic target for DLBCL, as inhibition of CXCR4 and tumor cell mobilization is bound to increase the accessibility of these lymphomas to anticancer thera- pies. Validating this strategy, the Food and Drug Administration-approved drug AMD3100 has been shown to potentiate the tumor growth inhibition afford- ed by standard chemotherapeutics and/or irradiation in preclinical models of glioblastoma,26,47 to inhibit the engraftment of B-cell acute lymphoblastic leukemia,48 to mobilize and sensitize acute promyelocytic leukemia cells to cytosine arabinoside,49 and to enhance the response to rituximab and to the anti-CD52 monoclonal antibody alemtuzumab in a mouse model of disseminat- ed lymphoma.50 In the clinical setting, encouraging pre- liminary data have been obtained from a phase II study using AMD3100 and standard chemotherapy in patients with acute myeloid leukemia.51 In DLBCL, pharmacolog- ical inhibition of CXCR4 by AMD3100 impairs the prop- agation of tumor B cells in a systemic mouse model of the
disease.12 Nonetheless, AMD3100 presents serious limita- tions that could preclude its use as an anticancer drug, such as some degree of cardiotoxicity and adverse phar- macodynamic properties including a positive charge at physiological pH and a limited half-life, impairing its oral bioavailability.52 Thus, the lack of a CXCR4 inhibitor suit- able for administration in a standard regimen supports the development of less toxic and more stable CXCR4- targeting agents.53
Here, we describe the biological activity of IQS- 01.01RS, a new potential CXCR4 inhibitor with a higher lethal dose in vivo and better pharmacodynamic proper- ties than AMD3100.35 We report that by interacting with a different CXCR4 domain than that with which AMD3100 interacts, this IQS-01.01RS acts as a putative allosteric CXCR4 inhibitor, impeding the activation of the receptor upon CXCL12 ligation and allowing a more sustained inhibition of the pathway. In vivo, this effect, together with better pharmacokinetic properties, was linked to an improved capacity of the compound to mobilize DLBCL cells. Furthermore, we show that IQS- 01.01RS-mediated blockade of CXCR4 signaling led to the post-transcriptional downregulation of MYC. Overexpression of this oncogene is associated with shorter survival in DLBCL patients,54 and is known to be stabilized by CXCR4 in cancer cells.55 MYC was consid- ered to be an undruggable factor until (+)-JQ1, a highly potent, selective and cell-permeable inhibitor of BRD4, a member of the BET family of chromatin adaptors, was first shown to have antitumor activity in multiple myelo- ma xenograft models.56 Further studies with (+)-JQ1 and CPI203, a molecule characterized by a superior bioavail- ability after oral or intraperitoneal administration,32,57 have validated BET bromodomain targeting and subse- quent blockade of MYC, NF-kB or BCL-2 protein family signaling as a promising therapeutic strategy in different subtypes of aggressive B-cell lymphoma, including DLBCL.58-60 BRD4 is a global regulator of gene transcrip- tion which selectively recognizes and binds to acetylated lysine residues in histones to activate transcription and mitosis. In DLBCL it preferentially localizes in super- enhancers associated with key transcription factors implicated in lymphomagenesis, such as MYC, which are specifically sensitive to BRD4 inhibition thus explain- ing the selective anti-tumor effect of BRD4 inhibitors.61 It is worth pointing out that BET bromodomain inhibition, as a consequence, downregulates other tumor-related genes apart from MYC.61 Nevertheless, in this work, we focused on the capacity of CPI203 to inhibit MYC, con- firming that ABC- and GCB-DLBCL cells are highly sen- sitive to CPI203 monotherapy, and that the transcription- al downregulation of MYC achieved by this compound allows almost complete abrogation of the protein when the compound is combined with IQS-01.01RS. This dual approach underlies the synergistic interaction of the two compounds and consequent sensitization to apoptosis in vitro and in vivo.
In conclusion, the present work shows that, besides CXCR4, the level of CXCL12 may also have an impact on the progression of DLBCL, and describes a new potent orally available CXCR4 inhibitor with antitumor proper- ties. In addition, this study offers the first rational basis for the potential clinical evaluation of a dual approach combining BET bromodain inhibition and CXCR4 block- ade in DLBCL.
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haematologica | 2019; 104(4)