IMiD screening methods IMiD-sensitive cells with mutant CRBN
The present invention features a knock-in mouse comprising a mutation in an endogenous CRBN locus and methods of use thereof.
1. A method of screening for an immunomodulatory imide drug (IMiD) that activates ubiquitin ligase, the method comprising:
a) contacting an isolated mouse cell with an IMiD, wherein the genome of the cell comprises an exogenous polynucleotide encoding a mutant cereblon (CRBN) polypeptide at the endogenous CRBN locus, wherein said mutant CRBN comprises the point mutation I391V, and wherein the cell has IMiD sensitivity; and
b) detecting the amount of:
i) ubiquitination of IKAROS family zinc finger protein 1 (IKZF1) or IKAROS family zinc finger protein 3 (IKZF3);
ii) degradation of IKZF1 or IKZF3; or
iii) binding of CRBN to IKZF1 or IKZF3,
in the isolated cell obtained in step a);
wherein increased ubiquitination or degradation of IKZF1 or IKZF3 or increased binding of CRBN to IKZF1 or IKZF3 as compared to a wild-type isolated mouse cell indicates the IMiD activates ubiquitin ligase.
2. The method of claim 1 , further comprising detecting:
i) ubiquitination of the proteome; or
ii) total protein level in the proteome, in the isolated cell of step a).
3. The method of claim 1 , wherein the immunomodulatory imide drug (IMiD) or agent is lenalidomide or an analog thereof.
4. The method of claim 3 , wherein the analog of lenalidomide is thalidomide or pomalidomide.
5. A method of screening for an immunomodulatory imide drug (IMiD) that activates ubiquitin ligase, the method comprising:
a) contacting an isolated mouse cell with an IMiD, wherein the genome of the cell comprises an exogenous polynucleotide encoding a mutant cereblon (CRBN) polypeptide at the endogenous CRBN locus, wherein said mutant CRBN comprises a point mutation selected from the group consisting of S369C, V380E, and I391V, and wherein the cell has IMiD sensitivity; and
b) detecting the amount of:
i) ubiquitination of IKAROS family zinc finger protein 1 (IKZF1) or IKAROS family zinc finger protein 3 (IKZF3);
ii) degradation of IKZF1 or IKZF3; or
iii) binding of CRBN to IKZF1 or IKZF3,
in the isolated cell obtained in step a);
wherein increased ubiquitination or degradation of IKZF1 or IKZF3 or increased binding of CRBN to IKZF1 or IKZF3 as compared to a wild-type isolated mouse cell indicates the IMiD activates ubiquitin ligase.
6. The method of claim 5 , further comprising detecting:
i) ubiquitination of the proteome; or
ii) total protein level in the proteome, in the isolated cell of step a).
7. The method of claim 5 , wherein the immunomodulatory imide drug (IMiD) or agent is lenalidomide or an analog thereof.
8. The method of claim 7 , wherein the analog of lenalidomide is thalidomide or pomalidomide.
9. An isolated mouse cell whose genome comprises an exogenous polynucleotide encoding a mutant cereblon (CRBN) polypeptide at the endogenous CRBN locus, wherein said mutant CRBN comprises a point mutation selected from the group consisting of S369C, V380E, and I391V, and wherein the cell has immunomodulatory imide drug (IMiD) sensitivity.