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Background <p>In chronic lymphocytic leukemia (CLL), B-cell receptor, chemokine receptor and integrin signaling contribute to disease progression by influencing cell survival, migration and microenvironment interactions. Focal adhesion kinase (FAK) has recently been involved in these disease processes. Our recent research revealed a correlation between activated FAK and molecules involved in CLL aggressiveness, particularly in IGHV-unmutated cases, including the Lyn kinase substrates HS1 and cortactin. In this context, ROR1 is a key player, sharing connections with Lyn substrates and FAK pathways.</p> Methods <p>We assessed FAK and ROR1 bidirectional association by stimulating CLL cells with Wnt5a and measuring FAK phosphorylation by Western blot, while ROR1 expression was evaluated following FAK inhibitor treatment. Flow cytometry using CXCR4 and CD5 markers was performed to identify and sort proliferating (CXCR4<sup>dim</sup>/CD5<sup>bright</sup>) versus quiescent (CXCR4<sup>bright</sup>/CD5<sup>dim</sup>) B-cell subpopulations from CLL patients. FAK and ROR1 levels were compared between these fractions. Findings were further supported ex vivo in ibrutinib-treated patients. The FAK inhibitor defactinib was tested alone and in combination with BTK inhibitors on primary CLL cells cultured with or without stromal support, measuring apoptosis by Annexin V/PI staining.</p> Results <p>We demonstrated that ROR1 triggering by Wnt5a increases FAK activation, while FAK inhibition reduces ROR1 expression. Significantly higher levels of FAK and ROR1 were detected in the proliferating subpopulation corresponding to cells egressing lymph nodes compared to quiescent cells. Ex vivo experiments confirmed high FAK and ROR1 levels in circulating lymphocytes redistributing from secondary lymphoid organs. Defactinib significantly enhanced apoptosis in CLL cells when combined with BTK inhibitors, even in supportive microenvironments, showing significant efficacy also against aggressive CLLs.</p> Conclusions <p>Our findings reveal a reciprocal regulatory relationship between FAK and ROR1, with both proteins enriched in CLL cells that have exited the lymph nodes. Given defactinib’s favorable safety profile in solid tumor trials, combining FAK inhibition with BTK inhibitors could enhance therapeutic efficacy by limiting leukemic clone adaptability and addressing resistance and relapse. While anti-ROR1 therapies showed limited efficacy as monotherapy in CLL trials, targeting multiple nodes of this regulatory network may prove more effective.</p>

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Focal adhesion kinase FAK interplays with ROR1 in the aggressiveness of chronic lymphocytic leukemia

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月27日

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  • Maria Castronuovo,
  • Guido Capasso,
  • Allison Beltrame,
  • Nayla Mouawad,
  • Vincenzo Gramegna,
  • Paolo Fantato,
  • Elisa Pagnin,
  • Francesco Angotzi,
  • Alessandro Cellini,
  • Andrea Serafin,
  • Monica Facco,
  • Andrea Visentin,
  • Livio Trentin,
  • Federica Frezzato

摘要

Background

In chronic lymphocytic leukemia (CLL), B-cell receptor, chemokine receptor and integrin signaling contribute to disease progression by influencing cell survival, migration and microenvironment interactions. Focal adhesion kinase (FAK) has recently been involved in these disease processes. Our recent research revealed a correlation between activated FAK and molecules involved in CLL aggressiveness, particularly in IGHV-unmutated cases, including the Lyn kinase substrates HS1 and cortactin. In this context, ROR1 is a key player, sharing connections with Lyn substrates and FAK pathways.

Methods

We assessed FAK and ROR1 bidirectional association by stimulating CLL cells with Wnt5a and measuring FAK phosphorylation by Western blot, while ROR1 expression was evaluated following FAK inhibitor treatment. Flow cytometry using CXCR4 and CD5 markers was performed to identify and sort proliferating (CXCR4dim/CD5bright) versus quiescent (CXCR4bright/CD5dim) B-cell subpopulations from CLL patients. FAK and ROR1 levels were compared between these fractions. Findings were further supported ex vivo in ibrutinib-treated patients. The FAK inhibitor defactinib was tested alone and in combination with BTK inhibitors on primary CLL cells cultured with or without stromal support, measuring apoptosis by Annexin V/PI staining.

Results

We demonstrated that ROR1 triggering by Wnt5a increases FAK activation, while FAK inhibition reduces ROR1 expression. Significantly higher levels of FAK and ROR1 were detected in the proliferating subpopulation corresponding to cells egressing lymph nodes compared to quiescent cells. Ex vivo experiments confirmed high FAK and ROR1 levels in circulating lymphocytes redistributing from secondary lymphoid organs. Defactinib significantly enhanced apoptosis in CLL cells when combined with BTK inhibitors, even in supportive microenvironments, showing significant efficacy also against aggressive CLLs.

Conclusions

Our findings reveal a reciprocal regulatory relationship between FAK and ROR1, with both proteins enriched in CLL cells that have exited the lymph nodes. Given defactinib’s favorable safety profile in solid tumor trials, combining FAK inhibition with BTK inhibitors could enhance therapeutic efficacy by limiting leukemic clone adaptability and addressing resistance and relapse. While anti-ROR1 therapies showed limited efficacy as monotherapy in CLL trials, targeting multiple nodes of this regulatory network may prove more effective.