Page 188 - Haematologica May 2020

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tion with SRF, regulates expression of hundreds of genes involved in cell migration, adhesion, and differentia- tion.15,39 We show that the high mRNA and protein expression of MKL1 in HL0 cells correlated with high expression of the MKL1 target genes SRF and ACTB. MKL1 activity promotes migration and proliferation of cancer cells,16,40,41 suggesting that HL0 LCL could be pre- malignant.
Because the absence of MKL1 results in a severely impaired actin cytoskeleton,19 we reasoned that overex- pression of MKL1 could result in a more active actin cytoskeleton. Actin cytoskeleton proteins are upregulated in invasive cancer,42 suggesting that the increased activity of the actin cytoskeleton in the HL0 cells could enhance cell migration and cell division, promoting invasion of cells and growth of tumors. In support of this, HL0 cells with the highest MKL1 expression had higher G- and F- actin content and displayed increased spreading, a process that depends on actin cytoskeleton dynamics. The migra- tion and hyperproliferation of cancer cells are also regulat- ed by alteration of integrin expression at the cell surface and modification of the subsequent intracellular integrin signaling.43 EBV-transformed B cells aggregate in cell cul- ture via the interaction between the integrin CD11a (a subunit of LFA-1) and the adhesion molecule ICAM-1 (this study).22-24 We examined LCL aggregation in vitro and observed that HL0 cells aggregated poorly, while HL1 cell aggregation varied and HL2 cell aggregation was similar to that of control cells. We found that the levels of surface and total protein as well as mRNA expression of CD11a were low in HL0 cells, but similar to control levels in HL2 cells. Interestingly, CD11a expression in HL1 cells showed a bimodal distribution with CD11a low and CD11a high cells, which might be the cause of the high variation of HL1 cell aggregation results. It is possible that the overac- tive actin cytoskeleton in HL0 cells prevented correct translocation of adhesion receptors to the cell surface or affected integrin inside-out and outside-in signaling.
Because of the possible pre-malignant stage of HL0 cells, we investigated cell proliferation in vitro and in vivo through formation of tumor mass. HL0 cells were hyper- proliferative in vitro and also in vivo where they rapidly formed tumors in immunocompromised NSG mice. The HL0 population showed increased DNA content and a higher percentage of hyperploid cells. Because polyploidy is a result of incomplete cytokinesis,44 the increased hyperploidy of HL0 cells could be due to an elevated rate of cytokinesis failure, possibly connected to overactivity of the actin cytoskeleton as shown previously when the actin regulator WASp is overactive.45,46 Reed-Sternberg cells originate from failed cytokinesis and re-fusion of HL daughter cells.47,48 This suggests that the failure of HL0 cells to complete cytokinesis could lead to the formation of giant multinucleated cells similar to Reed-Sternberg cells.
The variable phenotype of the HL1 and HL2 triplets treated for HL in 1985 and 2008, respectively, was at first puzzling. Since all triplets contained the heterozygous deletion of MKL1 intron 1, we reasoned that whether a cell expresses the healthy MKL1 allele or the intron 1- deleted allele of MKL1 must be a stochastic event. Transcription of the MKL1 gene is quite complex, with several sets of transcripts. The transcriptional start site is in exon 4, placing intron 1 in the 5’ untranslated region of MKL1. To understand the MKL1-associated phenotype,
we took advantage of the bimodal expression of CD11a in HL1 cells to sort out CD11a low and CD11a high cells. CD11a high cells behaved similarly to the control cells, displaying low expression of MKL1, low spreading and proliferation, together with a strong aggregation response. In contrast, CD11a low cells behaved similarly to HL0 cells with high expression of MKL1, decreased aggregation, as well as increased proliferation in vitro and tumor formation in vivo. We also sorted out the small pop- ulation of CD11a low cells from control (C1 and C2) cells; however, control CD11a low cells were not stable in cul- ture and started to express CD11a during 17 days of cul- ture. This suggests that the stable CD11a low phenotype in HL0 cells and sorted HL1 cells is associated with increased MKL1 expression. Interestingly, HL Reed- Sternberg cells are characterized by low mRNA expres- sion of CD11a.49,50 Thus, since the HL0 triplet has not received HL treatment we reason that HL0 cells could represent a pre-HL stage. The presence of CD11a low cells in HL1, who was treated for HL with mustargen, oncovin, procarbazine, prednisone/adriamycin, bleomycin, vinblastine, and dacarbazine in 1985,20 may indicate de novo HL with the CD11a low cells possibly outgrowing the healthy CD11a high cells. With this rea- soning, the HL2 patient who received adriamycin, bleomycin, vinblastine and dacarbazine to treat HL in 200820 should have predominantly CD11a high cells, assuming that the highly proliferative CD11a low cells would have been mostly eradicated by the HL treatment.
Finally, we investigated whether the HL0 cell pheno- type could be reverted by inhibition of MKL1 using the small molecule CCG-1423.17,18 HL0 cells treated with this MKL1 inhibitor showed decreased expression of MKL1 protein and the MKL1 target gene SRF, as well as reduced cell spreading. Importantly, using the MKL1 inhibitor we could revert the hyperproliferation of HL0 cells in vitro and also, by intratumoral injections, in vivo.
We present here the first example of an intron mutation in MKL1, resulting in increased MKL1 expression, increased actin content, decreased aggregation, hyperpro- liferation, and genomic instability in B cells. Of the triplets’ samples, those from HL0 showed the most pro- nounced difference in both MKL1 expression and activity, as well as in cellular responses when compared to con- trols. Cells from HL1 and HL2, successfully treated for HL, had a phenotype closer to that of healthy controls. This finding, together with the known role of MKL1 in metastasis15 and with high mRNA expression of MKL1 in many types of lymphomas, suggests that increased MKL1 expression actively participates in B-cell transformation and the pathogenesis of HL.
Acknowledgments
We are grateful to the family and healthy controls for providing blood samples. We thank Dr Kaisa Lehti, Dr Peter Bergman, and Dr Andreas Lundqvist for valuable input. This work was support- ed by a postdoctoral fellowship from the Swedish Childhood Cancer Fund and the Swedish Society of Medical Research to JR, an MD-PhD (CSTP) fellowship and a clinical internship (research AT) from Karolinska Institutet to AS, a clinical postdoc- toral fellowship from the Swedish Society of Medical Research to HB, a postdoctoral fellowship from Olle Engqvist Byggmästare to MH, a PhD fellowship from Fundação para a Ciência e a Tecnologia to MMSO, funds from the Swedish Medical Society to HB and LSW, Groschinsky Foundation and Åke Wiberg
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