P-200: MAP4K2 inhibition reinforces sensitivity to iberdomide in RAS-mutated MM through a CRBN-independent mechanism

Introduction: MAP4K2 is predominantly and highly expressed in the germinal center of B cells. We have shown that MAP4K2 knockdown in K- or N-RAS mutated MM cells inhibits cell growth associated with the downregulation of IKZF1/3, BCL-6, and c-MYC (Blood, 2021; 137:1754). Since IKZF1 regulates IRF4 and c-MYC transcription in MM, we hypothesized that IKZF1 degradation is one of the major mechanisms mediating MAP4K2-knockdown-induced MM cell death.

Methods: IKZF1 Q146H mutant, which abrogates E3 ligase cereblon (CRBN)-mediated IKZF1 degradation, was constructed by mutagenesis PCR. MM.1SRASmut cells were lentivirally transduced to overexpress IKZF1WT or IKZF1Q146H and treated by MAP4K2 inhibitor (TL4-12), lenalidomide, or iberdomide. IKZF1 degradation was detected by WB and MM growth inhibition by proliferation assay.
To investigate the combined effects of Iberdomide with MAP4K2 silencing on MM tumor growth, we generated subcutaneous MM xenografts in NOD/SCID mice using the inducible MAP4K2 shRNA MM.1SRASmut cells. MAP4K2 silencing was induced by doxycycline diet after the tumor was established.

Results: As expected, lenalidomide failed to induce IKZF1-degradation in IKZF1Q146H MM.1SRASmut cells, confirming that IMiDs-induced IKZF1 degradation is mediated by CRBN. In contrast, IKZF1Q146H did not protect MM.1SRASmut from MAP4K2 inhibitor TL4-12-induced IKZF1 downregulation. Proliferation assays confirmed that IKZF1Q146H MM.1S cells were resistant to LEN-induced cell growth inhibition but not to TL4-12, demonstrating that MAP4K2 inhibition causes IKZF1 degradation and cell growth inhibition through a mechanism different from that of lenalidomide. We further evaluated the effects of MAP4K2 silencing in IKZF1Q146H MM.1SRASmut cells. MAP4K2 knockdown resulted in significant inhibition of cell proliferation and increase of apoptotic cells in both IKZF1WT and IKZF1Q146H MM.1S. IKZF1WT or IKZF1Q146H were also overexpressed in IMiDs resistant (RPMI-8266RASmut) MM cell line. MAP4K2 knockdown showed similar effects on IKZF1 degradation, cell proliferation, and apoptosis in IKZF1Q146H compared to IKZF1WT RPMI8266RASmut MM cells, confirming that MAP4K2 inhibition induces anti-MM effects by degrading IKZF1 via a CRBN independent mechanism and subsequently overcomes IMiDs resistance.
Combination of iberdomide and MAP4K2-silencing led to synergistic in vitro anti-MM effects in RASMut MM cells. In our MM.1S xenograft mice model, combination of iberdomide with MAP4K2-silencing using a doxycycline-inducible shRNA approach further enhanced tumor inhibition and significantly prolonged mice survival compared to the single treatment.

Conclusions: Combination of MAP4K2 inhibition with Iberdomide results in synergetic anti-cancer effects in lenalidomide-resistant MM. Our findings demonstrate that MAP4K2 is a novel therapeutic target to bypass IMiDs resistance in RAS-mutated MM. Therefore, this approach will provide a strategy to manage relapsed or refractory patients with multi-drug resistance and RAS-mutated MM.