Revolutionizing B-ALL Cell Line Development: Novel Generation to Uncover Therapeutic Vulnerabilities
Authors: Wu Yiming , Szoltysek Katarzyna , Brown Tony , Backhaus Emily , Khan Tanya , Gao Qingsong , Brewer Amanda , Chang Yunchao , Roberts Kathryn G. , Crews Kristine R , Iacobucci Ilaria , Wang'ondu Ruth , Mullighan Charles G. 
Published: December 2024

B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer, with over 20 genomics subtypes. Cell line models are crucial for identifying vulnerabilities and developing precision therapies. However, most B-ALL cell lines are KMT2A-r, BCR::ABL1+, TCF3::PBX1 and MEF2D-r, leaving many subtypes unrepresented. Here, we developed a novel approach to generate B-ALL cell lines.

Previous methods have adapted patient samples directly into standard culture media such as RPMI1640+FBS with low success rates. Here, we initiated culture from frozen patient-derived xenograft (PDX) cells. These were thawed and cultured with human mesenchymal stromal cells (MSCs) in serum-free medium, SFEM II, generally promoting expansion within a week. We then assessed serum and stromal cell dependency and further adapted PDX cells to stromal-free culture either with or without FBS. A new cell line was defined if it underwent exponential expansion for at least eight passages without culturing with MSCs. The cell lines were then cyropreserved and characterized with short tandem repeat analysis and whole-genome/transcriptome sequencing (WGS/WTS).

We first developed and tested the workflow with nine low hypodiploid (LH) and seven BCR::ABL1-like PDX samples, including CRLF2-r (N=4) and ABL1-r, EPOR-r and NTRK3-r (N=1 each) samples sourced from the St. Jude PDX repository PROPEL (propel.stjude.cloud). In the initial step, 10 samples proliferated on MSCs in SFEM II medium. In the serum and stromal cell dependency test, we found BCR::ABL1-like samples either grew without MSCs in medium with FBS or co-culture with MSCs in SFEM II medium, while LH samples only proliferated in SFEM II medium either with or without MSCs. Overall, two to four months was required to generate a new cell line from PDX samples. In total, we generated 10 new B-ALL cell lines of 17 attempted, with seven having a doubling time of less than seven days.

We then tested the workflow with 17 PDX samples, including five NUTM1-r samples, four BCR::ABL1+ samples with either wild type or dominant negative (IK6) IKZF1 genotypes, two IKZF1 N159Y and TCF3::HLF samples, and one sample each with near haploid, PAX5alt, PAX5 P80R, and ZEB2/CEBPE. NUTM1-r samples showed the lowest success rate, with only one cell line generated. Eleven cell lines were generated from the other 12 PDX samples, with one IKZF1 N159Y sample failing. This demonstrated our approach has high success rate and is compatible with the majority of B-ALL subtypes. Notably, all these new cell lines consistently match the subtypes of PDX/original patient samples via t-SNE analysis, indicating they faithfully reflect the transcriptomic features of their corresponding subtypes. Additionally, we found BCR::ABL1+, BCR::ABL1-like, near haploid, and HLF-r samples grew in medium both with and without FBS, while other subtypes only grew in SFEM II medium. Notably, most samples required co-culture with MSCs or SFEM II medium, particularly subtypes not previously modeled, which in part explains the high success rate of this approach.

Finally, we studied the therapeutic vulnerabilities of five BCR::ABL1+/ BCR::ABL1-like samples with wild type IKZF1 or IK6 and five LH cell lines samples by testing cytotoxicity of 40 commercial drugs. LH lines showed resistance to many cytotoxic drugs, especially birinapant, inotuzumab, dexamethasone, and nelarabine. Consistent with previous publications, LH samples were sensitive to FLT3/PI3K, MEK, and BCL-2 inhibitors. Additionally, we found LH samples also respond well to vincristine and JQ1, suggesting new vulnerabilities. Surprisingly, BCR::ABL1+/BCR::ABL1-like samples with IK6 were sensitive to dexamethasone and gilteritinib compared to those with wild type IKZF1, though complete cell eradication was not achieved even at high dosage, indicating combined therapy would be beneficial.

In summary, we developed a new approach, generating 21 B-ALL cell lines spanning 11 subtypes. Combined with commercial lines, we now have seven LH lines, eight BCR::ABL1-like lines, five HLF-r lines, and four near haploid lines to further study the mechanisms and therapeutic vulnerabilities of these high-risk B-ALL subtypes. These models provide an important resource for identifying dependencies and therapeutic vulnerabilities.