Abstract 426: Ongoing TCRβ VDJ rearrangement in pre-T LBL cell lines

Abstract

Abstract The prognosis of recurrent precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL) is poor, mainly due to chemotherapy resistance. The specific mechanism(s) leading to chemoresistance at relapse is still poorly understood. We previously characterized a mouse strain that contains a CreERT2 cassette “knocked into” the 3′ UTR of Mcm2. Mice with 2 copies of this knock-in allele, designated Mcm2cre/cre show decreases Mcm2 protein expression. They die of pre-T LBL, due to acquired DNA copy number abnormalities (CNA), involving important tumor suppressor or oncogenes. We hypothesize this unique mutator phenotype may reveal specific genetic drivers leading to chemoresistance. We selected six drugs with different mechanisms that are known to be active against pre-T LBL, including cytarabine, etoposide, melphalan, methotrexate, prednisone, and vincristine. We used 4 mcm2 Pre-T LBL cell lines (#2883, 2696, 2641 and 2869). After 1 year of treatment, we obtained resistant cell lines that can tolerate drug concentrations which are up to 10,000X that of parental cell lines. These samples will be characterized by Sparse WGS (for CNA), RNA-Seq and WES. Before sequencing, we wanted to verify that cell lines had not become cross-contaminated during drug selection. During thymocyte differentiation, noncontiguous genomic V, D, and J gene segments become juxtaposed, and form an exon which encodes the variable region of a T cell receptor protein. The TCRβ VDJ rearrangement provides a unique identifier for pre-T LBL cell lines. For the 2869 cell line, we found that the VDJ sequences of cytarabine, vincristine, melphalan, and prednisone resistant 2869 were identical to the parental 2869 cells. However, analysis of 2883 cell lines were more complex. Although there were multiple VDJ rearrangements in parental 2883, they had an identical junction of D2J2S4, suggesting that the variable VDJ rearrangements were derived from a single clone with a unique DJ rearrangement, and subsequent different V-DJ rearrangements. Moreover, following drug selection, the VDJ rearrangement of chemo-resistant 2883 and parental 2883 were not identical. However, the chemo-resistant 2883 cells retain the fingerprint of parental 2883, namely the unique junction of D2J2S4. The VDJ rearrangement of etoposide resistant and vincristine resistant 2883 (2883ER and 2883VR, respectively) clones were identical, and could be identified as a subclone of parental 2883. Interestingly, the2883ER and 2883VR clones had nearly identical CNA of a number of genes, including Abcb1a (also known as Multi-Drug Resistance gene 1a), suggesting selection of a pre-existing resistant clone. Although the VDJ rearrangement of cytarabine 2883 was not found in the parental 2883 subclone, however, the unique D2J2S4 was present. These findings indicate that pre-T LBL cell lines do not invariably have stable TCRβ VDJ rearrangements, and can undergo V-DJ rearrangement event after several years of continuous cell culture. Citation Format: Dengchao Cao, Mianmian Yin, Toshihiro Matsukawa, Taimour Baslan, Peter Aplan. Ongoing TCRβ VDJ rearrangement in pre-T LBL cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 426.