Peptide-based antagonists for targeting CXCR4 in ibrutinib resistant chronic lymphocytic leukemia Free

Bruton tyrosine kinase inhibitors (BTKi) have revolutionized chronic lymphocytic leukemia (CLL) treatment. However, resistance develops, making it important to identify alternative treatment targets. CXCR4, a G protein coupled receptor, regulates stem cell homeostasis, lymphopoiesis and migration of immune cells. It promotes CLL proliferation by regulating the homing of CLL cells from peripheral blood to the pro-proliferative lymphoid organs. While BTKi are known to target CXCR4 signaling (Chen et al., 2016), the cycling of cells between the peripheral blood and lymphoid organs is re-established upon BTKi resistance. Therefore, targeting CXCR4 may represent a valuable treatment strategy in BTKi resistance.

Here, we studied the effects of the optimized derivatives of the Endogenous Peptide Inhibitor of CXCR4 (EPI-X4) (Harms et al., 2024) on human leukemia/lymphoma cell lines and murine models of ibrutinib resistance. Based on CXCR4 expression, and CXCL12 mediated transmigration and signaling, MEC2, HG3, JEKO1 and MAVER1 from a panel of 10 cell lines were selected for studying responses to EPI-X4 derivatives. Out of 6 tested peptides, the fatty acid-coupled JM#143, JM#194 and JM#198 showed binding to CXCR4 with IC50s of 14, 16 and 27nM compared to 575nM for plerixafor in JEKO1 cells. The EPI-X4 fatty acid conjugates inhibited CXCL12 mediated ERK and AKT phosphorylation at lower concentrations compared to plerixafor in MAVER1 (350-508nM vs. 2964nM), JEKO1 (634-721nM vs. >30µM), HG3 (160-248nM vs. 789nM) and MEC2 cells (78-92nM vs. 1346nM). For further studies the well characterized JM#198 peptide was used in comparison with plerixafor.

Resistance to ibrutinib was generated in the Eµ-TCL1 murine CLL model. In primary Eµ-TCL1 mice, CD5+ B cells expand in peripheral blood at 20 weeks of age, progressing to overt leukemia and death at a median of 50 weeks. We randomized N=20 animals aged 17-32 weeks for treatment with 30 mg/kg ibrutinib (N=14) or vehicle (N=6), administered as drinking water formulations. Treatments continued until the mice became sick. Median survival of the ibrutinib and vehicle treatment groups were 57 and 47 weeks, while the median duration of ibrutinib vs. vehicle treatments were 33 weeks (range: 6-47 weeks) vs. 24 weeks (range: 17-47 weeks), respectively. Splenocytes isolated from the ibrutinib and vehicle treated mice were analyzed ex vivo for CXCR4 expression and response to EPI-X4 derivatives.

CXCR4 expression was increased in Eµ-TCL1 tumors from ibrutinib treated mice compared to vehicle treatment and healthy B-cells from C57Bl6/J mice but the difference was not statistically significant. However, a negative correlation of CXCR4 expression with duration of treatment was observed in the ibrutinib treatment group. Ex vivo targeting of CXCR4 by EPI-X4 derivatives either as a single agent or in combination with ibrutinib did not affect cell survival after 4 days of treatment. However, JM#198 treatment led to a 10-fold reduction in transmigration towards CXCL12, similar to that of plerixafor in tumors from vehicle and ibrutinib treated mice. In comparison 1µM ibrutinib only marginally affected transmigration towards CXCL12.

Since ibrutinib treatment alters the T-cell microenvironment, we studied CXCR4 expression in the CD4 and CD8 T-cell subsets, and the impact of CXCR4 inhibition on T-cell function. Ibrutinib treatment normalized the expression of CXCR4 in effector (CD44^highCD127^low), memory (CD44^highCD127^high) and naïve (CD44^lowCD127^high) CD8+ T-cells to levels in healthy B-cells. Among CD4 subpopulations, the % of regulatory T cells (Tregs, CD25+ FOXP3+) were normalized to healthy B cell levels following ibrutinib treatment. In line with this, T-cells derived from ibrutinib treated mice showed increased CD25 and CD69 activation upon stimulation with anti-CD3 antibody in both the CD4+ and CD8+ T-cells. 24-hour treatment with CXCR4 antagonists did not alter the long term ibrutinib treatment mediated benefits on T-cell function.

In summary, the peptide antagonists of CXCR4 inhibit signaling in CLL/MCL cell lines. CXCR4 levels remained high in tumors derived from ibrutinib treated mice. Targeting CXCR4 using JM#198 strongly suppressed transmigration of tumor cells towards CXCL12 without adversely affecting T cell immunomodulation by ibrutinib. Combined targeting of CXCR4 and BTK could effectively block tissue homing of CLL cells, improving BTKi efficacy and potentially overcoming resistance.