L-540

Chromosome Instability in Hodgkin Lymphoma Cells Associated with Sister Chromatid Cohesion Defects

Aberrant sister chromatid cohesion causes chromosome instability (CIN) and thus contributes to the development of cancer. The study confirms that L-540 cell lines exhibit CIN phenotypes and are aneuploid. The L-540 cell lines were found to be aneuploid compared to the diploid control. The total chromosome numbers ranged from 41 to 148, with a model chromosome number of 56, and normal chromosomes ranging from 56 to 74.

Cohesion normally functions by tethering nascent synthesized chromatids together to prevent premature segregation and thus chromosome instability. Cohesion was categorized into normal (cohesed) or aberrant (primary constriction gap; PCG). PCG is a visually appreciable gap between the sister chromatids within the DAPI channel. To further characterize the severity of the PCG phenotypes, all spreads exhibiting aberrant cohesion were further classified into three distinct sub-categories ranging from mild (PCG_I) to moderate (PCG_II) to severe (PCG_III). In L-540, the cohesion was 44.6%, while PCG_I, PCG_II, and PCG_III were 14.9%, 10.6%, and 29.9%, respectively (Fig. 1).

Fig. 1 Representative images of mitotic chromosome spreads from the L-540 cells. (Sajesh BV, et al., 2013)

Aberrant Expression of HLX in Hodgkin Lymphoma

NKL homeobox genes regulate cell and tissue differentiation. The HLX gene, encoded by NKL, exhibits abnormal activity in patients with Hodgkin's lymphoma (HL) cells. Here, genomic profiling (Fig. 2A) and fluorescence in situ hybridization (Fig. 2B) were performed to examine if HLX is abnormally activated in L-540.

To identify potential upstream factors of HLX, expression profiling of L-540 was conducted compared to HL cell lines. The results indicated activation of JAK-STAT signaling with high statistical significance. To examine directly the impact of STAT factors, the study quantified expression levels and performed siRNA-mediated knockdown of STAT5A, STAT5B, STAT3, and STAT4 (Fig. 3). STAT3 knockdown resulted in decreased HLX transcript levels, demonstrating that STAT3 activated the expression of HLX (Fig. 3C).

Overall, the study investigations excluded chromosomal rearrangements of the HLX locus at 1q41 (Fig. 2A) and demonstrated that STAT3 operated directly as a transcriptional activator of the HLX gene (Fig. 3). These analyses indicate that aberrantly expressed NKL homeobox gene HLX is part of a pathological gene network in HL, driving deregulated B-cell differentiation and survival.

Fig. 2 (A) Genomic profiling was performed for seven HL cell lines, including L-540 cell lines. (B) Fluorescence in situ hybridization (FISH) in L-540 demonstrated wild-type configurations at the locus of HLX although chromosome 1 showed some structural abnormalities. (Nagel S, et al., 2018)

Fig. 3 RQ-PCR analysis of STAT-factors in HL cell lines, including L-540 cell lines. (Nagel S, et al., 2018)

Lestaurtinib Inhibiting Proliferation and Inducing Apoptosis in Hodgkin Lymphoma

JAK/STAT constitutive activation plays an important role in the pathogenesis of HL. Lestaurtinib is an orally bioavailable multi-kinase inhibitor recently shown to inhibit JAK2. The study analyzed the effect of Lestaurtinib treatment in L-540 cell lines.

Proliferation and apoptosis in response to Lestaurtinib of cultured HL cells were evaluated in L-540 cell lines after 48 h of treatment and compared to cells treated with DMSO vehicle control (normalized to 100%). At 300 nM of Lestaurtinib, a 66% reduction in proliferation was observed in L-540. At 300 nM of doxorubicin, the reduction in proliferation was 54% in L-540 (Fig. 4). At 300 nM, apoptosis increased by 10% in L-540. No significant differences were observed between 48, 72, or 96 hours.

The JAK/STAT pathway is one of the most frequently altered pathways in HL. To assess in greater detail the effects of Lestaurtinib-mediated JAK2 inhibition on the JAK2/STAT5 signaling pathway, protein levels of STAT5, phosphor-STAT5, STAT3, and phosphor-STAT3 were then analyzed. Following 1 hour of 300 nM of Lestaurtinib treatment, phosphor-STAT5, and phosphor-STAT3 levels decreased respectively (Fig. 5). After 1 h of 300 nM of Lestaurtinib treatment, Bcl-xL mRNA expression levels had decreased by 37% in L-540 (Fig. 6). This downregulation of Bcl-xL could explain the proapoptotic effect of Lestaurtinib.

Fig. 4 Proliferation (left) and apoptosis (right) analysis after 48 h of Lestaurtinib treatment in L-540 cell lines. (Diaz T, et al., 2011)

Fig. 5 Western blot analysis of JAK2/STAT5 pathway protein levels in L-540 cells after 1 h of Lestaurtinib treatment at different doses: 30, 100, and 300 nM. (Diaz T, et al., 2011)

Fig. 6 Bcl-xL mRNA analysis after 1 h of Lestaurtinib treatment in L-540 cell lines. (Diaz T, et al., 2011)