Cereblon (CRBN) Splicing Could Influence Response To IMiDs : A New PCR Strategy to Easily Detect and Semi-Quantify Loss Of The IMiDs Binding Domain
Authors: Ménard Audrey , Touzeau Cyrille , Lok Anne , Amiot Martine , Pellat-Deceunynck Catherine , Maiga Sophie , Magrangeas Florence , Minvielle Stephane , Blin Nicolas , Mahe Beatrice , Dubruille Viviane , Tessoulin Benoit , Halliez Maxime , Wuillème Soraya , Robillard Nelly , Moreau Philippe , Maisonneuve Hervé , Jardel Henry , Bene Marie C. , Moreau Philippe , Lodé Laurence 
Published: October 2019

Abstract
Cereblon (CRBN) was recently identified as a protein binding immunomodulatory drugs (IMiDs) (Ito et al., 2010, Lopez-Girona et al., 2011) essential for the activity of thalidomide, lenalidomide and pomalidomide in patients suffering from multiple myeloma (MM) (Zhu et al., 2011 & 2012). It is known that some patients develop resistance to these drugs, raising the question of a possible role of CRBN alterations (mutations, deletions, loss of transcription...) in the development of such treatment escapes (Broyl et al., 2012, Heintel et al, 2013).
Ito et al (2010) showed that loss of the C-ter fragment of CRBN or missense mutations (Y384A and W386A) in the thalidomide binding domain (TBD) indeed impair the efficacy of this drug.
We investigated whether the loss of CRBN expression, particularly of full length isoforms including exons 10 and 11 encoding TBD, or missense mutations could be detected by polymerase chain reaction (PCR) followed by fragment-length analysis or Sanger bidirectional sequencing. This method was applied to a cohort of 19 patients issued from a consecutive cohort of 45 elderly patients treated with lenalidomide and dexamethasone for relapsed or refractory MM (Touzeau et al., 2012).
RNA was extracted from plasma cells purified by CD138 immunomagnetic sorting (STEM CELLS® beads) at the time of diagnosis. Sanger sequencing performed on RT-PCR products revealed no missense mutations but disclosed a high frequency of alternative splicing of CRBN in samples from MM patients (Lodé et al 2013). We thus developed a new PCR strategy to detect and semi-quantify alternative spliced isoforms of CRBN involving or not a loss of CRBN specific domains, focusing on exons 10 and 11. Two PCR were performed with custom-made primers: PCR-1 was designed to encompass the whole coding sequence of CRBN and PCR-2 was specifically designed to cover TBD. Fragment-length analysis of fluorescent PCR-2 products was obtained by capillary electrophoresis on an Applied Biosystems 3130xl Genetic Analyser (Applied Biosystems, Foster City, CA) allowing for precise sizing and semi-quantification by assessing the height of peaks.
This PCR strategy was highly efficient to detect both unspliced CRBN transcripts (PCR-1 and PCR-2 fragments detected at 1562 nucleotides (nt) and 568nt, respectively) and CRBN alternatively spliced variants. The sizing strategy chosen allowed to identify exon skipping. For instance, PCR-2 fragments without exon 10 were visualized as peaks of 234nt, 319nt and 436nt depending on the presence or not of exons 7, 8 and 9.
Alternative CRBN splicing in TBD was seen in nearly all patients of this cohort. Examples of representative splicing patterns are shown in figure 1. Semi-quantification of CRBN spliced transcripts showed that 58% of patients had lost more than 50% of CRBN full-length isoform and that 37% had lost more than 50% of exon10. Of note, two patients with a very short duration of response to lenalidomide and dexamethasone (16 days and 58 days respectively) had lost more than 95% of full-length CRBN and more than 90% of exon 10 (see MM6179 in figure1) in initial samples in spite of proper detection of an actin fragment of 858nt.Figure1Figure1.
The fragment-length PCR analysis presented here demonstrates that CRBN splicing profiles in MM patients are very heterogeneous. A correlation with response to treatment was not established in this rather small cohort of patients, but this could be due to the fact that diagnosis samples were collected too early in relation to the time of initiating lenalidomide treatment. Indeed, CRBN splicing could be therapy-induced.
A prospective study is needed to determine whether the semi-quantification of CRBN alternatively spliced variants would indeed correlate with, or even predict, clinical response to IMiDs. Moreover, further mutation studies are needed to elucidate the heterogeneity of splicing profiles which could reflect the presence of subclonal mutations in genes encoding the splicing machinery.

Disclosures:
Moreau: CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau.