Deciphering the IKZF1 and MAP4K2 protein interactome in RASMut multiple myeloma reveals a novel regulatory pathway of IKZF1 Free

Background: RAS pathway mutations are common in multiple myeloma (MM) and linked to MAP4K2 upregulation, a potential therapeutic target (Li et al., Blood 2021). Inhibiting MAP4K2 in RAS^Mut MM impairs cell growth and downregulates IKZF1/3, BCL-6, and c-Myc. Our prior work showed MAP4K2 inhibition triggers IKZF1 degradation independently of CRBN, the known E3 ligase for IKZF1 and IMiD mediator. Here, we explore the IKZF1 interactome and its CRBN-independent regulation to identify novel strategies for promoting IKZF1 degradation as a therapeutic approach in MM.

Methods: TurboID proximity labeling was used to map IKZF1 and MAP4K2 interactomes in MM. IKZF1-TurboID and nuclear TurboID (NLS-TurboID) were stably expressed in RAS^Mut MM.1S cells, treated with MAP4K2 inhibitor BAY-61-3606 or CELMoD mezigdomide (positive control), then biotin-labeled. MAP4K2-TurboID and cytosolic TurboID (NES-TurboID) were expressed in MM.1S cells. Biotinylated proteins representing interacting proteins were enriched by streptavidin beads and analyzed by LC-Mass-Spectrometry (MS).

To functionally complement the interactome mapping, a CRISPR-Cas9 BISON library screen (Słabicki et al., Nature 2020) was performed in MM.1S cells expressing mCherry-IKZF1, treated with BAY-61-3606 or mezigdomide. FACS-sorted mCherry^high cells underwent sgRNA sequencing to identify ubiquitin E2/E3 ligases and DUBs regulating IKZF1. CRISPR data were analyzed using Galaxy Australia (Nucleic Acids Res 2024), LC-MS data with Python, and Pathway/TRRUST analyses with Metascape (Zhou et al., Nat Commun 2019).

Results: LC-MS analysis identified 1,533 proteins significantly enriched in IKZF1-TurboID samples over NLS-TurboID controls in MM. Our assay confirmed known IKZF1 interactors such as transcriptional factors (IKZF3/4 and FOXK1/2), HDAC complex members (HDAC2, MTA1/2 and GATAD2A/B) and SUMOylation-associated proteins (PIAS1/2/4), which were previously reported in HEK293 cells using a less sensitive IKZF1-BioID assay (Astori et al., J Immunol. 2020). Compared to the IKZF1 interactome in HEK293 cells, our data revealed additional transcription factors including MYC, JUN and FOS as well as key signaling and stress response proteins TRAF5, DUSP1, TP53BP2, CD70 and MAP3K1. Furthermore, in the context of the proteasome-ubiquitin system, our TurboID analysis uncovered a broader set of interactors, including TRIM ligases and Culin-Ring ligases. These findings highlight the cell-type-specific interactome of IKZF1 and underscore its distinct regulatory roles in MM.

Mezigdomide, a cereblon E3 ligase modulatory (CELMoD), induces IKZF1 degradation via high-affinity CRBN binding and serves as control for drug-induced IKZF1 interactome. As expected, CRBN was enriched explicitly in the mezigdomide-treated IKZF1-TurboID pulldowns, but not in MAP4K2 inhibitor BAY-61-3606-treated samples.

BAY-61-3606 treatment in IKZF1-TurboID pulldown enriched 112 proteins, mainly involved in mRNA processing, ribonucleoprotein biogenesis, and alternative splicing, which are predominantly regulated by SIRT1 and TP53. Top enriched proteins included CCSER1, ICAM1, HIC1, FOXK1, CCNT1, AUTS2, STRBP, FOXO3, and HRNPUL1, suggesting MAP4K2 may reprogram the myeloma transcriptome. Additionally, we identified 7 ubiquitin E3 ligases and 2 DUBs within this dataset as well as NF-κB regulators. In the comprehensive CRISPR-KO screening assay, CRBN, DDB1, and RBX1 were the top hits in mezigdomide-treated samples, validating this approach. Several candidate E3 ligases and DUBs, including NF-κB and NEDDylation pathways regulators, were involved in MAP4K2 inhibition-induced IKZF1 degradation. These candidates are currently being validated.

MAP4K2-TurboID interactome identified 625 proteins enriched over the control, linked to RNA metabolism, cell division, and viral infection pathways, and regulated by MYCN, DNMT1, and TP53. The top hits included ALG8, NAA30, MAP4K3, FOXK1, and GSK3B. Notably, 16 MAP4K2 interactors, including CCNT1, MORC3, FOXK1, NOC2L, RB1, RASA2, and NKRF, also showed increased binding to IKZF1 upon MAP4K2 inhibition, suggesting MAP4K2 directly regulates the IKZF1 transcriptional complex.

Conclusions: This study presents the first IKZF1 interactome map in MM using TurboID, coupled with a CRISPR-based functional screen, and suggests new pathways such as NF-κB for IKZF1 degradation. Identification of the MAP4K2-dependent IKZF1 degradation mechanism offers a promising strategy to overcome IMiD resistance.