IKZF1/3 and CRL4^CRBN E3 Ubiquitin Ligase Mutations Associate with IMiD Resistance in Relapsed Multiple Myeloma

Immunomodulatory drugs (IMiDs) provide the backbone for various Multiple Myeloma (MM) treatment regimens. Cereblon (CRBN) is the known key modulator of the IMiD anti-tumor effects as it is the intermediate protein within the CRL4^CRBN E3 ubiquitin ligase (CRL4) complex that targets the degradation of the two transcription factors, Ikaros (IKZF1) and Aiolos (IKZF3). We hypothesize that the formation of thiscomplex (CRBN-DDB1-CUL4B-ROC1) is essential for the IMiD's action and that mutations that occur in involved genes affect its assemblage, thus induce IMiD resistance.

Targeted sequencing was performed using the M3P (v2.0 or v3.0) gene selection. Average sequencing depth was 700X. We have combined data from different cohorts analyzed with the M3P for a total of 501 patients, 337 newly diagnosed and 164 pretreated. We also included data from the CoMMpas trial (804 newly diagnosed MM analyzed by WES with 100X average depth).

We analyzed a total of 1.305 MM cases and identified 53 mutations in 42 patients within CRBN, IKZF1, IKZF3 and CUL4B . Mutation frequency increased significantly after treatment (Z-score: 6.9, p<0.001), with 1.9% in untreated (22/1141) and 12.2% in (20 /164) pretreated cases. Of note, this analysis does not include either DDB1, one of the binding partners of CRBN in the complex, nor ROC1, in the pretreated cohort, as it was not part of the M3P gene selection. In untreated patients, however, mutation incidence was low with 4 out of 804 (0.5%) for DDB1 and no mutation was identified in ROC1 within patients included into the CoMMpass trial.

CRBN was the most commonly mutated gene within the investigated CRL4 complex genes (21 mutations /15 MM patients), followed by CUL4B (15/13). Of note, in IKZF3 (11/11) and IKZF1 (6/6) we identified two new hotspots (IKZF3 p.G159R/A and IKZF1 p.A152T, two patient each). Furthermore, five of the patients harbored mutations in IKZF1/3 within 30 amino acids previously described to be essential for IMiD sensitivity in vitro (Krönke et al., Science, 2014). 91% (20/22) of the CRBN and CUL4B mutations found in treated patients were nonsense or were located within close proximity to the binding sites of the proteins (Figure), causing loss/weakening of lenalidomide (LEN)-CRBN, CRBN-DDB1, CUL4B-DDB1 or CUL4B-ROC1 interactions. This includes a patient having progressed on Thalidomide and Pomalidomide therapy with parallel evolution of 7 subclonal mutations in CRBN, 3 located in the LEN binding and 4 in the DDB1 binding site. Response data were available for 19 out of 20 treated patients with mutations in the CLR4 complex and, strikingly, 18 (95%) of them were clinically unresponsive to IMiD treatment.

In summary, CLR4 mutations are predominantly selected by therapy and correlate with IMiD resistance. In our cohort of pretreated patients the incidence of CLR4 mutations was 12%, thus we provide evidence of a significant role of single point mutations as an IMiD drug resistance mechanism in MM.

Figure: Mutations in CRL4^CRBN E3 ubiquitin ligase clustered in the binding sides of complex unitsA) Mutations close to the binding side of Lenalidomide. B) Mutations in the interface CRBN-DDB1 and C) DDB1-CUL4B. D) Mutations in the binding side of ROC1. The structure of the CRL4 complex was produced by the alignments LEN-CRBN-DDB1 (PDB: 4CI2) and DDB1-CUL4B-ROC1 (PDB 4A0C). CRBN CTD (LEN binding) domain is shown in gray and HBD (DDB1 binding) in plun, LEN in orange, DDB1 in sky blue, CUL4B in pink and ROC1 in yellow.

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