Combination of JAKi and HDACi Exerts Antiangiogenic Potential in Cutaneous T-Cell Lymphoma

Abstract

Angiogenesis plays a pivotal role in the growth and metastasis of tumors, including the development and progression of cutaneous lymphomas. The chick embryo CAM model has been utilized in various studies to assess the growth rate, angiogenic potential, and metastatic capability of different tumor types and malignant cell lines. However, the precise mechanisms of angiogenesis in CTCL and the influence of Ruxolitinib or Resminostat on angiogenesis in hematological malignancies and solid tumors are not well understood. Recent in vitro and in vivo data have demonstrated the synergistic inhibition of tumorigenesis and metastasis in experimental models of CTCL when using the combination of Resminostat (HDACi) with Ruxolitinib (JAKi). The present work aims to elucidate the effects of this combination on the tumor microenvironment's vascular components. We investigated the effects of Ruxolitinib (JAKi) in combination with Resminostat (HDACi) treatment in transendothelial migration of CTCL cells (10⁶ MyLa and SeAx) by using a transwell-based transendothelial migration assay and tumor angiogenesis in vivo. We used the CTCL chick embryo CAM model with xenografted tumors derived from implanted MyLa and SeAx cells and administered topically 15 μM ruxolitinib and 5 μM Resminostat every two days during a 5-day period. JAKi and HDACi inhibited CTCL cell transendothelial migration by 75% and 82% (p < 0.05) in both CTCL engrafted cells (MyLa and SeAx, respectively) compared to the untreated group. Moreover, the combination of ruxolitinib with resminostat blocked angiogenesis by significantly reducing the number of blood vessel formation by 49% and 34% in both MyLa and SeAx, respectively (p < 0.05), indicating that the proposed combination exerted significant anti-angiogenic effects in the CAM CTCL model. Overall, these data provide valuable insights into potential therapeutic strategies targeting angiogenesis in CTCL, paving the way for more effective treatment approaches in the future.

Keywords: CTCL; angiogenesis; chick embryo model; resminostat; ruxolitinib.

Figure 1

Ruxolitinib/Resminostat inhibited transendothelial migration in MyLa and SeAx engrafted cells in chick embryo model. Values represent mean ± standard error of the mean (SEM) for three independent experiments. Welchs’ t-test was implemented for comparison of two independent groups. One-way ANOVA analysis with Welch correction was performed for multiple comparison tests. Significance was defined as p ≤ 0.05 and denoted as: ** p < 0.01 **** p < 0.001, ns: not significant. Analysis was performed using GraphPad Prism 8 software (San Diego, CA, USA).

Figure 2

Ruxolitinib/Resminostat blocked angiogenesis in MyLa (A) and SeAx (B) engrafted cells in chick embryo model. Representative images of (A) patterns of vascular branching and IKOSA blood vessels map (left panel) and its quantification (right panel) in grafted CTCL microtumors into the CAM treated as indicated. Arrows in the image indicate the effect of drugs on blood vessel density and integrity. Combination of ruxolitinib and resminostat resulted in the development of thin and collapsed blood vessels with reduced size lumens compared to control. Values represent mean ± SEM for four independent experiments, each employing 10–12 embryos per treatment variant. * p < 0.05, ** p < 0.01, *** p < 0.005, ns: not significant by one-way ANOVA test.

Figure 3

Expression levels of angiogenesis-related genes in MyLa/SeAx engrafted cells in chick embryo model after treatment with resminostat and/or ruxolitinib. Combination treatment decreased VEGFA (A) and increased VEGFB (B) in CTCL cells engrafted in chich embryos compared to vehicle. The bars are the means determined in three (n = 3) independent experiments using 10–12 embryos per variant. * p < 0.05, ** p < 0.01, *** p < 0.005, ns: not significant by one-way ANOVA test.

Figure 4

Ruxolitinib/Resminostat decreased hemoglobin levels in MyLa (A) and SeAx (B) engrafted cells in chick embryo model compared to vehicle. The bars are the means determined in three (n = 3) independent experiments using 10–12 embryos per variant. * p < 0.05, ** p < 0.01, **** p < 0.001 by one-way ANOVA test.

Figure 5

Ruxolitinib/Resminostat decreased tumor mass in MyLa (A) and SeAx (B) engrafted cells in chick embryo model compared to vehicle. Data show mean ± SEM from three (n = 3) independent experiments, each employing 12–14 embryos per treatment variant. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001 by one-way ANOVA test.

Figure 6

HDAC gene expression and HDAC activity in MyLa (A) and SeAx (B) engrafted cells in chick embryo model. Combination of Ruxolitinib/Resminostat and resminostat alone decreased HDAC activity levels in MyLa and SeAx engrafted cells in chick embryo model. Data show mean ± SEM from three (n = 3) independent experiments, each employing 10–12 embryos per treatment variant. ** p < 0.01, *** p < 0.005 , ns: not significant by one-way ANOVA test.

Figure 7

Western blot analyses in CTCL CAM onplant microtumors for key implicated pathways. Portions of microtumors were lysed and analyzed in MyLa (A) and SeAx (B) CAM onplant microtumors for the activation levels p-AKT (Ser473), p-ERK (Tyr 204), p-STAT5 (Tyr694/699), and GADPH in . Data show mean ± SEM from three (n = 3) independent experiments. p values: * < 0.05, ** < 0.01, *** < 0.005, **** < 0.001 by one-way ANOVA test.