Page 180 - Haematologica May 2020

J. Record et al.
a transcriptional coactivator of serum response factor (SRF) and binds to globular (G-)actin via an RPEL motif.7,8 As cyto- plasmic G-actin is polymerized into filamentous (F)-actin, the G-actin pool diminishes. This leads to MKL1 transloca- tion into the nucleus where it interacts with SRF to induce transcription of cytoskeleton-related genes, including actin, integrin molecules, and SRF itself.7-10 Indeed, inducible expression of SRF in response to serum stimulation is dependent on SRF and MKL1 activity.9,11 Actin polymeriza- tion and MKL1-SRF activity are additionally regulated by extracellular signaling through several integrin molecules which activate the small Rho GTPases, including RhoA.12
MKL1 was initially described as part of a fusion protein in megakaryoblastic leukemia of poor prognosis.13,14 MKL1 expression is detected in malignant cells in breast and liver cancer and is associated with increased cell proliferation, anchorage-independent cell growth, and metastasis.15,16 Small molecule inhibitors of the MKL1-SRF pathway have been identified, facilitating studies on the biological activity of MKL1, and are being tested as potential cancer therapeu- tic agents.17 One of these compounds is CCG-1423, which was originally identified as a RhoA-MKL1-SRF pathway inhibitor and later discovered to target MKL1 directly.17,18
A loss-of-function mutation in MKL1 was recently iden- tified in a 4-year old girl with severe primary immunodefi- ciency.19 MKL1 deficiency caused reduced G-actin and F- actin content in the patient’s neutrophils, leading to reduced phagocytosis and migration.19 In 2013, a familial case of two monozygotic triplets who developed HL at the age of 40 and 63 was described.20 Both patients are in remission fol- lowing HL treatment in 1985 and 2008, respectively, and the third triplet remains undiagnosed. Using microarray comparative genomic hybridization, a 15-31 kb deletion in intron 1 of MKL1 was identified in the triplets.20 The impact of this mutation on MKL1 expression and B-cell function remains unknown.
Here we took the approach of generating EBV-trans- formed lymphoblastoid cell lines (LCL) from the triplets with the deletion in MKL1 intron 1 (HL0, HL1, and HL2) and from two healthy controls (C1 and C2). We found that the LCL from the undiagnosed triplet had increased MKL1 and SRF expression, and elevated G-actin content. This was associated with hyperproliferation, genomic instability, and tumor formation when the cells were injected into immunocompromised mice. When compared to control LCL with high CD11a expression and capacity to form large aggregates, HL0 LCL expressed low CD11a and had reduced capacity to form aggregates. The HL1 LCL showed a bimodal expression of CD11a and when sorted for CD11a low and CD11a high cells, CD11a high cells mim- icked the response of control LCL whereas the H10 CD11a low cells mimicked the response of HL0 cells with increased proliferation and tumor formation. Finally, treat- ment of HL0 cells with the MKL1 inhibitor CCG-1423 reverted the phenotype and prevented tumor growth in vivo. These data show that unregulated MKL1 alters B-cell cytoskeletal responses leading to B-cell transformation.
Methods
Human blood samples and Epstein-Barr virus transformation
Whole blood samples were obtained from the triplets and age-matched controls after informed consent was given. This
study was performed according to the principles expressed in the Helsinki Declaration and with approval from the local ethics committee (Dnr 2015/416-31). For analysis of primary cells, the first experiment included samples from HL0, HL1, and a control (not used for EBV-transformation) collected in February 2015 and the second experiment was conducted on samples from HL2 and C1, collected in May 2015. To establish the EBV-trans- formed LCL, peripheral blood mononuclear cells from HL0, HL1, and HL2, and two age- and sex-matched controls (C1 and C2), all collected in November 2015, were cultured with super- natant of the virus-producing B95-8 line.21
Mice
Flow cytometry and microscopy
Flow cytometry was performed on peripheral blood mononu- clear cells, LCL, and cultured primary B cells using an LSRFortessa X-20 (BD Biosciences). The results were processed using FlowJo v10 software (TreeStar Inc., St. Ashland, OR, USA). To determine integrin expression at the cell surface, LCL were labeled with an anti-human CD11a antibody (TS2/4; Biolegend) for total CD11a expression, or an anti-human CD11a antibody (Hl111; Biolegend) for inactive/closed conformation-CD11a expression, or an anti-human CD54 antibody (Biolegend), fol- lowed by an anti-mouse-Alexa647 antibody (ThermoFisher Scientific). To determine F- and G-actin content in two LCL sam- ples side by side, one sample was incubated with an anti-human CD54 antibody (Biolegend) for 30 min on ice and thereafter labeled with DNaseI-Alexa488 (ThermoFisher Scientific) and phalloidin-Alexa568 (ThermoFisher Scientific).
Results
The MKL1 intron 1 deletion is associated with increased expression of MKL1 and MKL1-induced genes
To understand how the deletion in MKL1 intron 1 affected actin cytoskeleton regulation in B cells, we exam- ined freshly isolated cells and LCL from the triplets (HL0, HL1, and HL2) and two healthy controls (C1 and C2) (Figure 1A, B). We reasoned that cells from the undiag- nosed HL0 triplet may be in a pre-HL stage, whereas HL1 and HL2 cells may be more similar to control cells because of successful treatment for HL in 1985 and 2008, respectively. MKL1 protein in primary blood lympho- cytes was higher in the cells from all triplets than in con- trol cells, as assessed by flow cytometry (Figure 1C and Online Supplementary Figure S1A). Using primer walking and sequencing, we confirmed that the triplets’ cells con- tained a heterozygous deletion of MKL1 intron 1 (Online Supplementary Figure S2A). The intron 1 is in the 5´ untranslated region of the MKL1 gene. We examined exon boundaries of exons 1-4 in the 5’ untranslated region and found normal expression of adjacent exons,
NOD/SCID-IL2rγnull (NSG) mice were bred and maintained at the animal facility of the Department of Microbiology, Tumor and Cell Biology at Karolinska Institutet under specific pathogen-free conditions. Female mice were used and all animal experiments were performed after approval from the local ethics committee (the Stockholm District Court, permit N77/13 and N272/14). For inhibitor treatment, 10 mM CCG-1423 or dimethylsulfoxide was injected intratumorally for 6 consecutive days. The volume of the tumor was calculated at the endpoint using a caliper.
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