Inhibition of the Integrin αVβ3 Improves the Immune and Anti-Lymphoma Effects of Bexarotene in Cutaneous T-Cell Lymphoma (CTCL)
Authors: Cayrol Maria Florencia , Debernardi Maria Mercedes , Revuelta Maria Victoria , Paulazo Alejandra , Calvo Vidal Maria Nieves , Phillip Jude M , Sterle Helena , Diaz Flaque Maria Celeste , Cremaschi Graciela , Cerchietti Leandro 
Published: April 2021

Abstract
Bexarotene (Bex) is an oral RXR agonist that is effective for the treatment of early and advanced-stage CTCL. However, Bex is associated with the unavoidable side effect of hypothyroidism in about 95% of pts, which is prophylactically managed with the administration of thyroid hormone (TH). Paradoxically, we found that physiological levels of TH increase the proliferation of CTCL by activating both the nuclear TRA receptor and the membrane integrin αVβ3 in these cells. Here, we determined the influence of TH replacement therapy on the anti-lymphoma activity of Bex, an unknown topic with clinical implications.
Results: In standard culture conditions, Bex decreases the proliferation and viability of CTCL cell lines HuT78 and MJ. We conducted RNA-sequencing in HuT78 cells treated with Bex (vs. vehicle) to understand the mechanism/s underpinning these effects. We found that Bex regulates pathways related to "cell proliferation and differentiation" (e.g. REL, SCD, CCND1) and "immune system" (e.g. TBX21, IFNG, MX1) , which we independently validated. This suggests that Bex treatment could also impact anti-neoplastic immunity by increasing INF-gamma release from CTCL cells. We then conducted the same experiment culturing HuT78 and MJ in TH-depleted culture conditions and observed a higher effect of Bex in decreasing proliferation and viability; supporting the notion that Bex should not be administered with TH replacement. However, hypothyroidism is associated with marked immunosuppression that could favour CTCL progression and potentially affect the immune modulator effect of Bex that we described above.
We thus determined the impact of TH replacement on the anti-lymphoma effect of Bex using a murine CTCL model. We implanted murine EL4 TCL cells in the hypodermis of immunocompetent C57BL/6 mice. Once tumors developed, mice were randomized into treatment with vehicle (Veh), Bex with no TH replacement (Bex) and Bex with TH replacement (BexT4+). TH replacement was done with oral administration of T4. We measured T4 in these mice plasma by RIA to assure that mice with T4 replacement reach physiological levels and those with no T4 develop hypothyroidism. Compared to Veh mice, the administration of Bex significantly decreased lymphoma growth in both conditions although slightly better in mice without T4 replacement (p&lt;0.001 and p&lt;0.01) for Bex and BexT4+, respectively). However, mice with Bex alone that developed hypothyroidism showed a significant decrease of CD8+CD44+ T-cells (p&lt;0.05 vs. BexT4+) and increase of myeloid-derived suppressor cells in the tumor microenvironment and draining lymph nodes. Since infiltration of CD8+ cells decreases with CTCL progression, our data indicates that Bex treatment should be administered with T4 replacement to avoid a negative immune microenvironment.
Considering the cellular effects of TH are exerted through TRA and integrin αVβ3 that can be independently modulated pharmacologically, we investigated whether integrin αVβ3 inhibition would be sufficient to blunt the TH-induced decrease on the antineoplastic efficacy of Bex. The lack of expression of integrin αVβ3 in normal T-cells offers a rationale for a selective effect on CTCL cells while sparing immune cells. In HuT78 and MJ cell lines, either αVβ3 silencing by siRNAs or pharmacologic inhibition with the clinical drug cilengitide avoided the decrease in the anti-CTCL effect of Bex upon TH supplementation. Moreover, gene expression analysis (RNA-seq and PCR) demonstrated that αVβ3 silencing induced higher (vs. Bex) up-regulation of "immune " genes like TBX21 and INFG, or down-regulation of "proliferation" genes like REL and CCND1 .
We thus tested if this mechanism can be therapeutically capitalized to improve Bex treatment in our CTCL murine model. We implanted 24 C57BL/6 mice with murine EL4 TCL cells and randomized them to vehicle (Veh), Bex with TH replacement in combination with cilengitide 40mg/kg (BexT4+Cil) or without cilengitide (BexT4+). At end of treatment, BexT4+Cil mice showed significantly smaller tumors compared with Veh and BexT4+ mice (p&lt;0.001 and p&lt;0.05, respectively). Importantly, the anti-tumoral immune response of CD8+CD44+ T-cells in the CTCL microenvironment and draining lymph nodes remained similar in BexT4+Cil vs. BexT4+ mice. Our data indicates that inhibition of the integrin αVβ3 is an effective strategy to improve Bex-based treatments in CTCL.

Disclosures
Cerchietti: Celgene: Research Funding; Lymphoma Research Foundation: Research Funding; Leukemia and Lymphoma Society: Research Funding; Weill Cornell Medicine - New York Presbyterian Hospital: Employment.