IQGAP1 is a novel non-degraded substrate of CELMoDs and involved in trafficking of immune cells to the tumor microenvironment Free

Introduction: Cereblon E3 ligase modulating drugs (CELMoDs) represent a novel class of immunomodulatory agents that target IKZF1 and IKZF3 for degradation, building upon earlier generations such as Thalidomide and Avadomide. CELMoDs have demonstrated potent direct cytotoxicity and efficacy in hematologic malignancies, including multiple myeloma and lymphoma. RNAseq and multiplex immunofluorescence analyses of paired clinical biopsies (screening and on-treatment) from diffuse large B-cell lymphoma (DLBCL) patients treated with Avadomide revealed increased infiltration of lymphocytes and myeloid cells into the tumor microenvironment (TME). Similar findings were observed in patients treated with Golcadomide (Golca), a next generation CELMoD currently under clinical investigation for DLBCL, follicular lymphoma, and T cell lymphoma, and were recapitulated in a genetically engineered humanized cereblon-DLBCL mouse model, highlighting the translational significance of CELMoD-induced immune modulation. However, the molecular mechanisms driving CELMoD-mediated leukocyte trafficking remain incompletely understood. IQGAP1, a multidomain scaffolding protein, is essential for cellular migration, accumulating at the leading edge of migrating cells and interacting with activated Rac and CDC42 to facilitate actin cross-linking to generate protrusive forces. Here, we present evidence that IQGAP1 is recruited to CRBN in the presence of Golca and investigate the molecular mechanisms underlying Golca mediated enhancement of leukocyte trafficking.

Methods: Ubiquitinomics profiling was conducted using automated CST PTM-SCAN enrichment of K-GG peptides, followed by LC-MS/MS, and AI/ML-based peptide identification and quantification. Ubiquitin immunoprecipitation (IP) was performed using anti-multi-ubiquitin mAb-magnetic beads. CRISPR/Cas9 knockouts of either IQGAP1 or cereblon (CRBN) were generated in OCI-AML2 monocytic cells via lentiviral transduction. Protein–protein interactions were assessed using Duolink® proximity ligation assays (PLA) in primary human monocytes and human-CRBN mouse spleen tissues, employing CRISPR knockout-validated antibodies for in situ and FFPE staining. Trans-endothelial migration assays were performed in Boyden chambers to evaluate cell trafficking to chemokine gradients.

Results: IQGAP1 was identified as an additional protein band sequenced from Thalidomide bead pulldown experiments using cell lysates treated with Avadomide. Subsequent proteomic analyses confirmed that IQGAP1 co-IP with CRBN in the presence of various CELMoDs. Ubiquitin IP revealed enhanced ubiquitination of IQGAP1 in cells treated with Golca, while total IQGAP1 protein levels remained unchanged and did not undergo proteasomal degradation, suggesting that ubiquitination serves a regulatory function. Ubiquitinomics profiling further showed that Golca treatment significantly increased ubiquitination at three specific lysine residues on IQGAP1—K953, K1445, and K1475—which are located within the GRD-RGCT domains associated with Rac/CDC42 interactions.

PLA assays showed that Golca mediated a significant over three-fold increase in CRBN:IQGAP1 and IQGAP1:CDC42 interactions in both primary human monocytes and in spleens from hCRBN DLBCL mouse models. Functional migration assays further demonstrated that Golca mediated a significant over two-fold increase in chemotactic capacity toward a chemokine gradient using both primary human monocytes and OCI-AML2 cells. Disruption of either IQGAP1 or CRBN via CRISPR knockout in OCI-AML2 cells abrogated this enhancement, indicating that both CRBN and IQGAP1 are indispensable for Golca's effect on migration.

Collectively, these results reveal that Golca plays a pivotal role in driving enhanced leukocyte trafficking through the CRBN–IQGAP1 axis.

Conclusion: This study uncovers a novel mechanism by which Golca binding to CRBN mediates enhanced ubiquitination of three specific lysine residues on IQGAP1. This modification increased IQGAP1's interaction with Rac, CDC42 and actin, thereby promoting directional immune cell migration. Our findings provide the first description of IQGAP1 as a non-degraded CELMoD substrate, directly linking its biological function to clinical observation of increased immune cell infiltration into TME. These insights not only deepen our understanding of CELMoD action but also highlight the potential of CELMoD-based combination therapies to amplify the efficacy of immunotherapies.