RS4;11

The RS4;11 Cell Line as a Model for Leukaemia With t (4; 11) (q21; q23)

Hematological malignancies harboring rearrangements of the KMT2A gene represent a unique subtype of leukemia, with phenotypic clinical manifestations, a rapid and aggressive onset, and a generally poor prognosis. Chromosomal translocations involving KMT2A often cause the formation of oncogenic fusion genes, such as the most common translocation t (4;11) (q21; q23) producing the KMT2A-AFF1 chimera. This study aimed to confirm and review the cytogenetic and molecular features of the KMT2A-rearranged RS4;11 cell lines.

The presence of the t (4; 11) (q21; q23) was confirmed by M-FISH (Fig. 1), from which both der (4) and der (11) could be identified. The involvement of the KMT2A gene was further confirmed by FISH using a dual-color break-apart probe encompassing the KMT2A locus at 11q23.3. One normal KMT2A allele could be seen as a yellow fusion signal, whereas disruption of the KMT2A region would result in one red and one green signal present on the der (11) and der (4), respectively (Fig. 3).

Arm-specific probes showed the complete coverage of one chromosome 7 in red, corresponding to the long arm (Fig. 2C). For single locus probes, this was confirmed by the presence of two additional signals for 7q22 and 7q36 on one chromosome 7 (Fig. 2D). M-FISH (Fig. 1) and FISH using WCP8 (Fig. 2E) revealed two copies of chromosome 8 different in size and centromere position. FISH using a single locus DNA probe for 8q24.3 (RP11-195E4) was carried out to investigate a possible duplication of this locus. Signals specific to this region were visible on opposite sides of the centromere, suggesting the presence of an isochromosome 8q (Fig. 2F). The dual-color probe XL CDKN2A/2B highlighted the presence of both chromosome 9 centromeres. However, the lack of both signals for 9p21 revealed a homozygous deletion of that region (Fig. 2G, H).

Fig. 1 Representative karyotype of RS4;11 obtained by M-FISH. (Ragusa D, et al., 2019)

Fig. 2 Cytogenetic abnormalities investigated by FISH in the RS4;11 cell line. (Ragusa D, et al., 2019)

Fig. 3 Dual-colour FISH shows the KMT2A rearrangement in RS4;11. (Ragusa D, et al., 2019)

Differential Responses to SYK Inhibition in Precursor B-All Cells

The spleen tyrosine kinase (SYK) represents a potential therapeutic target in a subset of B-cell neoplasias. The biological and molecular effects of the SYK inhibitor entospletinib (Ento) in pre-B-ALL (NALM-6) and pro-B-ALL cell lines (SEM and RS4;11) were evaluated.

Ento exposure on pre-B-ALL NALM-6 (pre-BCR⁺) showed a concentration and slightly time-dependent decrease in cell proliferation. Similarly, Ento exposure also exhibited anti-proliferative effects on the pro-B-ALL cell line SEM (pre-BCR⁻). In contrast, the pro-B-ALL RS4;11 (pre-BCR⁻) showed only a moderate reduction in proliferation at the highest concentration of 10 µM and 20 µM applied for 48 h and 72 h (Fig. 4). High Ento concentrations induced significant early and late apoptosis/necrosis in the pre-B-ALL NALM-6 and SEM cell lines. In contrast, pro-B-ALL RS4;11 was almost resistant to apoptosis induction by Ento (Fig. 5). Cell-cycle distribution was investigated in three cell lines after Entospletinib exposure. Both concentrations of Ento (low concentration 1 µM or high concentration 10 µM) were not able to induce changes in cell-cycle distribution after 72 h incubation in the tested B-ALL cell lines (Fig. 6).

Fig. 4 Cell viability after entospletinib exposure in RS4;11 cell lines. (Sender S, et al., 2021)

Fig. 5 Apoptosis induction in RS4;11 cells after entospletinib (Ento) exposure. (Sender S, et al., 221)

Fig. 6 Flow cytometric cell-cycle analysis of the RS4;11 cells after entospletinib exposure. (Sender S, et al., 2021)