Phospholipase c gamma 2 and resistance associated mutations

Abstract

Described herein is a mutation that confers resistance to the treatment with a BTK inhibitor. Described herein is a modified PLCγ2 polypeptide that is modified at amino acid position 742, 845, or 1140 and the modified PLCγ2 polypeptide exhibits decreased inhibition (e.g., resistance) to a covalent and/or irreversible BTK inhibitor. Described herein are diagnostic methods for detecting the modified polypeptide and nucleic acid encoding the modified polypeptide and applications of the methods thereof. Described herein are compositions, combinations, and kits containing the modified polypeptide and methods of using the modified polypeptide. Also described herein are methods of using the modified polypeptide as screening agents for the identification and design of inhibitors of PLCγ2.

1 . A method of assessing whether a subject having a hematologic cancer is less responsive or likely to become less responsive to therapy with a BTK inhibitor, comprising:
 a. testing a sample containing a nucleic acid molecule encoding a PLCγ2 polypeptide from the subject;   b. determining whether the encoded PLCγ2 polypeptide is modified at an amino acid position corresponding to amino acid position 742, 845, or 1140 of the amino acid sequence set forth in SEQ ID NO: 2; and   c. characterizing the subject as resistant or likely to become resistant to therapy with a BTK inhibitor if the subject has the modification at amino acid position 742, 845, or 1140.   
     
     
         2 . The method of  claim 1 , wherein the subject has been administered a BTK inhibitor for treatment of a hematologic cancer. 
     
     
         3 . A method of maintenance therapy in a subject having a hematologic cancer, comprising:
 a. administering to the subject a maintenance therapy regimen comprising administering a therapeutically effective dose of a BTK inhibitor; and   b. monitoring the subject at predetermined intervals of time over the course of the maintenance therapy regimen to determine whether the subject has mutation in an endogenous gene encoding PLCγ2 that results in a modification at an amino acid position corresponding to amino acid position 742, 845, or 1140 of the amino acid sequence set forth in SEQ ID NO: 2.   
     
     
         4 . The method of  claim 1 , wherein the modification comprises a substitution, an addition or a deletion of the amino acid at amino acid position 742, 845, or 1140 in the PLCγ2 polypeptide. 
     
     
         5 . The method of  claim 4 , wherein the modification is:
 a. a substitution of asparagine to an amino acid selected from among leucine, cysteine, isoleucine, valine, alanine, glycine, methionine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, glutamine, aspartic acid and glutamic acid at amino acid position 742 of the PLCγ2 polypeptide;   b. a substitution of leucine to an amino acid selected from among cysteine, isoleucine, valine, alanine, glycine, methionine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 845 of the PLCγ2 polypeptide; or   c. a substitution of aspartic acid to an amino acid selected from among leucine, cysteine, isoleucine, valine, alanine, glycine, methionine, serine, threonine, phenylalanine, tryptophan, lysine, arginine, histidine, proline, tyrosine, asparagine, glutamine, aspartic acid and glutamic acid at amino acid position 1140 of the PLCγ2 polypeptide.   
     
     
         6 . The method of  claim 4 , wherein the modification in the PLCγ2 polypeptide is selected from among R742P, L845F, and D1140G. 
     
     
         7 . The method of  claim 1 , wherein the nucleic acid encoding the modified PLCγ2 polypeptide has a mutation of adenine to thymidine at B4823764.3 nucleic acid position corresponding to nucleic acid position 2535 in the sequence of nucleotides set forth in SEQ ID NO: 1. 
     
     
         8 . The method of  claim 1 , wherein the PLCγ2 polypeptide further comprises modifications at additional amino acid positions. 
     
     
         9 . The method of  claim 1 , further comprising discontinuing treatment with the BTK inhibitor if the subject has one or more modifications with at least one modification at amino acid position 742, 845, or 1140 in the PLCγ2 polypeptide. 
     
     
         10 . The method of  claim 1 , further comprising administering an inhibitor of PLCγ2 if the subject has one or more modifications with at least one modification at amino acid position 742, 845, or 1140 in the PLCγ2 polypeptide. 
     
     
         11 . The method of  claim 1 , wherein the subject possesses high-risk cytogenetic features. 
     
     
         12 . The method of  claim 11 , wherein the high-risk cytogenetic features comprise del(11q22.3), del(17p13.1) or complex karyotype. 
     
     
         13 . The method of  claim 1 , further comprising testing a sample containing a nucleic acid molecule encoding a PLCγ2 polypeptide and an additional polypeptide and determining whether the additional polypeptide contains mutations. 
     
     
         14 . The method of  claim 13 , wherein the additional polypeptide is a BTK polypeptide. 
     
     
         15 . The method of  claim 13 , wherein the testing is by isothermal amplification or polymerase chain reaction (PCR). 
     
     
         16 . The method of  claim 1 , wherein the BTK inhibitor is ibrutinib. 
     
     
         17 . The method of  claim 1 , wherein the hematologic cancer is a B-cell malignancy. 
     
     
         18 . The method of  claim 17 , wherein the B-cell malignancy is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-celllymphoma (DLBCL), activated B-cell diffuse large B-celllymphoma (ABC-DLBCL), germinal center diffuse large B-celllymphoma (GCB DLBCL), double-hit diffuse large B-celllymphoma (DH-DLBCL), primary mediastinal B-celllymphoma (PMBL), non-Hodgkin lymphoma, Burkitt's lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, precursor B-cell acute lymphoblastic leukemia, hairy cell leukemia, mantle cell lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma/Waldenstr6m macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. 
     
     
         19 . A system of detecting a modified PLCγ2 that confers resistance to inhibition with an irreversible BTK inhibitor in a subject, comprising:
 a. a sample containing a nucleic acid molecule encoding a PLCγ2 polypeptide from the subject; and 
 b. a microarray comprising nucleic acid encoding a modified PLCγ2 polypeptide or a portion thereof that is modified at an amino acid position corresponding to amino acid position 742, 845, or 1140 of the amino acid sequence set forth in SEQ ID NO: 2. 
 
     
     
         20 . The system of  claim 19 , wherein the microarray further comprises comprising nucleic acid encoding a modified PLCγ2 polypeptide or a portion thereof that is modified at additional amino acid positions.