Page 185 - Haematologica May 2020

Intronic deletion in MKL1 alters B-cell function
was seen between multinuclearity and DNA content (Figure 4E, F), indicating that increased DNA content could have resulted from genomic instability. When arresting LCL from the triplets and controls in metaphase, HL0 and HL1 had higher proportions of cells (approximately 25–35%) with more than 46 chromo- somes (Figure 4G, H). To investigate the growth of HL0 and C1 cells in vivo, the cells were injected subcutaneous- ly into NSG mice that lack a functional immune response and cell growth was examined as tumor mass on day 9. C1 cells failed to form a tumor mass whereas HL0 cells formed tumors with distinct angiogenesis (Figure 4I). Together these observations suggest that increased MKL1 expression and activity is directly associated with hyperproliferation and genomic instability leading to for- mation of a tumor mass in vivo.
HL1 cells contain two distinct populations of which one shows an HL0 phenotype
We noticed that HL1 cells recurrently showed an inter- mediate phenotype when compared to HL0 and control cells. HL1 cells showed a bimodal expression of CD11a and we examined whether this represented two popula- tions of cells with different phenotypes. We used fluores- cence activated cell sorting (FACS) to sort out CD11a low and CD11a high cells from the HL1 cells (Figure 5A). PCR of genomic DNA showed that sorted HL1 CD11a low and HL1 CD11a high cells contained the heterozygous deletion of MKL1 intron 1 (Online Supplementary Figure S2A). CD11a low cells had increased MKL1 protein and increased cell spreading when compared to CD11a high cells (Figure 5B, C). We quantified aggregation over 2 h and found that CD11a low cells had a reduced capacity to aggregate com- pared to that of CD11a high cells (Figure 5D, E). To exam- ine proliferation, we sorted 250,000 CD11a low and CD11a high cells and counted them between days 3 and 8. The CD11a high cells showed only modest proliferation whereas the CD11a low cells expanded 3- to 4-fold in the period between day 3 and day 8 (Figure 5F). To examine proliferation of cells in vivo, we injected CD11a low and CD11a high cells into NSG mice and determined tumor growth on day 15. CD11a low cells formed a large tumor mass with visible angiogenesis, whereas CD11a high cells formed a smaller tumor mass (Figure 5G). To address whether the control C1 and C2 cells also contained a stable CD11a low population, we used FACS and cultured the C1 and C2 CD11a low and CD11a high cells for 17 days. The C1 and C2 CD11a low cells gained expression of CD11a during the culture, suggesting that the CD11a phenotype in control cells was unstable (Online Supplementary Figures S7 and S8). We next examined whether alterations in CD11a expression were present in the L1236 HL cell line derived from a primary isolate of a patient with advanced HL.29 The L1236 cells contained large Reed-Sternberg-like cells and smaller cells (Figure 5H). Using flow cytometry, small and large cells were identified based on forward and side scatter. Small cells had low expression of CD11a and the large cells were devoid of CD11a expression (Figure 5H). Together, these data suggest that low expression of CD11a may be a characteristic feature of pre-malignant B cells in HL.
Inhibition of MKL1 activity in HL0 cells induces a phenotype similar to that of control cells
We next investigated whether we could revert the phe- notype of HL0 cells to that of control cells using the
MKL1 small molecule inhibitor CCG-1423.17,18 To define the dose range, HL0 cells were treated with different doses of CCG-1423 and cell death measured by flow cytometry. At doses of 2-10 mM CCG-1423, 90% of cells were viable (Figure 6A). HL0 cells treated with CCG- 1423 displayed dose-dependent lowering of MKL1 pro- tein and decreased SRF expression (Figure 6B-D). The spreading capacity of HL0 cells was reduced upon CCG- 1423 treatment (Figure 6E). Lowering MKL1 protein and activity led to increased aggregate formation and reduced proliferation (Figure 6F-H). To examine whether the MKL1 inhibitor could suppress tumor growth in vivo, HL0 cells were injected into NSG mice by subcutaneous injec- tions. From day 6 to day 12, a daily dose of 10 mM CCG- 1423 was injected intratumorally. Compared to treat- ment with vehicle (dimethylsulfoxide), treatment with the MKL1 inhibitor led to reduced HL0 tumor mass in mice (Figure 6I).
Discussion
Dysregulation of MKL1 expression and the actin cytoskeleton has been implicated in hematologic malig- nancies, although the exact mechanism has not been determined (Online Supplementary Figure S9).30,31 Mutated MKL1 was originally described in patients with acute megakaryoblastic leukemia in whom MKL1 was fused with RBM15 by a chromosomal translocation.13,14 The role of MKL1 in megakaryocyte differentiation, migration, and in the formation of proplatelets was subsequently described in MKL1-deficient mice.32,33 In humans, MKL1 deficiency has a profound affect on the hematopoietic cell actin cytoskeleton resulting in severely impaired cell migration and phagocytosis.19 Genetically identical triplets with a large deletion in MKL1 intron 1, of whom two affected by HL and one undiagnosed, provided a unique opportunity to investigate MKL1 and B-cell responses in the pathogenesis of HL. We found that increased activity of MKL1 in B cells led to the classical hallmarks of cancer cells: hyperproliferation, genomic instability, and formation of tumors with induction of angiogenesis.34
In this study we mainly used EBV-transformed B cells (LCL) from two controls and the triplets. To minimize possible variation due to EBV transformation, all samples were EBV-transformed under the same conditions with regards to day of transduction, virus batch and concentra- tion. The EBV cells enabled an extensive investigation of the impact of the MKL1 intron 1 deletion on B cells and importantly they allowed us to perform controlled exper- iments in which cells from the controls and the triplets could be compared side by side. A limitation with EBV transformation is that highly proliferative B cells are favored during EBV transformation.35 Moreover, the donor distribution of naïve, non-switched memory, and switched memory B cells is reflected in the EBV-trans- formed B cells.35,36 We cannot exclude that the EBV trans- formation induced expansion of a dominant subclone; however, CD11a staining revealed variable expression of CD11a in each sample although with different distribu- tions. In fact, the variable CD11a expression allowed us to define different subpopulations in the HL0 and HL1 samples and argue against a dominant subclone upon EBV transformation.
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