13 August 2018

Paper of the month July 2018

T315I mutation of BCR-ABL1 into human Philadelphia chromosome-positive leukemia cell lines by homologous recombination using the CRISPR/Cas9 system

Philadelphia chromosome arises as a feature of a translocation – change of parts – between two segments of Chromosomes 9 and 22 - t(9;22) - and allows for joining of two exons (coding part of the gene) existent in each of this chromosome’s parts(https://www.cancer.gov/images/cdr/live/CDR533336-750.jpg). BCR and ABL1 exons fuse, generating a transcript which codes for an abnormal synthesis of a tyrosine kinase, responsible for promoting uncontrolled cell growth. This leads to pathologies such as chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). Tyrosine kinase inhibitors (TKIs) like imatinib are administered in order to induce remissions in CML and Ph+ ALL patients. However, resistance to imatinib has been identified due to point mutations in the BCR-ABL1 exon like the T315I, which is mutated in its kinase domain, being of utmost importance to address it. One way is by establishing cell lines resistant to imatinib harboring this specific mutation and developing targeting strategies to overcome resistance to TKIs of first generation like imatinib and second-generation such as nilotinib and dasatinib. In order to prevent additional resistance mechanisms due to long-term exposure of cell lines to imatinib, a new system able to introduce T315I mutation in cell lines sensitive to imatinib is necessary.

In this sense, CRISPR/Cas9 technology is able to introduce mutations into cell lines using a homologous-recombination (HR) mechanism, where single-stranded oligodeoxynucleotides (ssODNs) can be provided to the cells together with Cas9 protein and sgRNAs homologues to the DNA sequence to be targeted (See paper April and May 2018: https://imgene.ku.dk/paper-of-the-month/). Leukemia cells such as Ph+ ALL show impairment in repair pathways of DNA damage and in order to introduce CRISPR/Cas9 system into these same cell lines, HR pathway must be functionally active.

In this paper, the authors use 4 Ph+ ALL cell lines resistant to Poly (ADP) ribose polymerase I (PARP1) inhibitors like olaparib (determined by IC50 measurements) which is translated by having an intact HR pathway. The objective of the work carried out was to introduce T315I mutation on the BCR-ABL1 transcript into these cell lines by HR-mediated CRISPR/Cas9 pathway that may serve as model of disease resistance to imatinib therapy. Ph+ ALL cells were electroporated with recombinant Cas9 protein together with 4 combinations of sense or anti-sense ssODNs with a point mutation of ACT to ATT at codon 315 - T315I mutation - and forward or reverse sgRNAs. Cells were cultured in the presence of imatinib after 7 days in order to establish specific resistance. Following this period, sublines of three imatinib-resistant Ph+ myeloid cell lines (K562, TCCS and KOPM28) harbored the T351I mutation as confirmed by PCR and enzyme digestion of PCR products and direct sequencing. The combination of reverse sgRNA and anti-sense ssODN showed the highest HR efficiency. Mainly, T315I-acquired sublines of K562, TCCS and KOPM28 cells showed constitutive phosphorylation of CRKL protein, which represents one of the downstream enzyme of BCR-ABL1 and no cell cycle arrest and apoptosis induction upon imatinib treatment was observed, resembling the resistance to imatinib. Treatment with dasatinib and nilotinib also didn’t affect T315I sublines viability, by opposition to parental cell lines. Ponatinib, a potent TKI, decreased viability of the three cell lines, being however possible to distinguish TCCS and KOPM28 sublines from K562 cells behavior regarding drug-response curves. Although these sublines had been established with T315I mutation in BCR-ABL1, K562 cells also acquired this mutation in ABL1 allele, which might explain the difference in drug-response to TKIs.

We recommend reading this paper in the sense that CRISPR/Cas9 envisions a new system to efficiently incorporate T315I mutations in three different leukemia cell lines to test drug sensitivities to TKIs, where the sublines resistant to imatinib established constitute useful cell line models for this purpose, as suggested by the authors.