The SKI proto-oncogene enhances the in vivo repopulation of hematopoietic stem cells and causes myeloproliferative disease

Abstract

© F e r r a t a S t o r t i F o u n d a t i o nat B.R.C. St Vincent's Hospital, Australia. All experiments were performed under the ethical guidelines of the Lund University or St Vincent's Health Melbourne animal ethics committees. 0.1-5x10 5 unsorted transduced cells from C57Bl/6 mice (BalbC in Online Supplementary Figure S3) were injected intravenously (IV) with 1-2x10 5 fresh whole BM cells into lethally irradiated recipients (8-12 weeks old, tracked using CD45.1/CD45.2). Cells from multiple transductions (n>8 for MIG and SKI; n=3 for ARPG) were transplanted into 4-6 recipients/transduction. Recipients were sacrificed after 16 weeks and the equivalent of half a femur of BM was transplanted into secondary and tertiary recipients. One cohort of mice was kept for 18 months. Cell preparations and flow cytometryPeripheral blood (PB), BM and spleen were analyzed on a Sysmex (Boule Medonice CA 530-16, or Sysmex KX-21N, Roche Diagnostics, Australia). FACS analysis was performed on a FACS Calibur or LSRFortessa, while cell sorting was performed on a FACS Diva or FACS Aria (BD, San Jose, CA, USA). Results were analyzed with FlowJo software, v.8.8.6 (Tree Star, Ashland, OR, USA). Hematopoietic progenitor assaysTo analyze TGF-β sensitivity, 200 GFP+ cells/mL were seeded in methylcellulose (M3231, Stem Cell Technologies), supplemented with 50 ng/mL mSCF, 10 ng/mL mIL-3, and 10 ng/mL hIL-6, with or without 10 ng/mL TGF-β (all from PeproTech, Rocky Hill, NJ, USA) (n=2 transductions). Unsorted BM cells from 16 weeks post-transplantation were plated for colonies; 30,000 cells/mL as above for myeloid and 50,000 cells/mL for pre-B-cell colonies (M3630). For HGF-analysis 250 sorted GFP+ cells/mL were seeded in methylcellulose with 50 ng/mL mSCF, 10 ng/mL mIL-3, 50 ng/mL hIL6, and 3 U/mL rhEpo (Jansen-Cilag) together with 0, 100 nm or 300 nm of MET Inhibitor III (Calbiochem cat. n. 448105) (n=3 separate transductions). All colonies were scored on Day 7. MorphologyPB smears and BM cytospins were stained with May Grünwald (Merck) and Giemsa (BDH). Megakaryocytes were scored in paraffin-embedded sections of decalcified tibiae stained with Mayer's hematoxylin and eosin (n=4 for MIG; n=5 for SKI). Images were taken using an S. Singbrant et al. 648 haematologica | 2014; 99(4) © F e r r a t a S t o r t i F o u n d a t i o nOlympus BH2 microscope and a MagnaFire-SP Optronics Digital Camera. Gene expression profilingQuantification of RNA was made on GFP+ BM cells using the Taq-Man™ System (Applied Biosystems, Foster City, CA, USA). For microarray, total RNA was extracted from GFP+ LKS cells using the RNeasy Micro kit (Qiagen), and hybridized to the Illumina Mouse WG-6 v.2.0 Expression BeadChip at the Ramaciotti Centre for Gene Function Analysis, University of NSW, Australia (n=6 independent transductions). For Illumina data analysis see the Online Supplementary Methods, differentially expressed genes in Online Supplementary Appendix File 1, enriched gene sets in Online Supplementary Appendix File 2, and raw and processed mi...

SMAD3 Determines Conventional versus Plasmacytoid Dendritic Cell Fates

“…In hematopoietic cells, SKI represses retinoic acid receptor signalling (Dahl et al, 1998), which enhances SMAD3/SMAD4-driven transactivation (Pendaries et al, 2003). SKI induces a gene signature associated with hematopoietic stem cells (HSCs) and myeloid differentiation, as well as hepatocyte growth factor (HGF) signalling (Singbrant et al, 2014). These previous reports and our finding suggest the possibility that HGF signals via STAT3 (Schaper et al, 1997) might induce synergy with c-SKI to repress SMAD3 toward myeloid differentiation.…”

“…SKI and the closely related SnoN oncogenes act as transcriptional corepressors in TGF-β signalling through interaction with SMADs (Akiyoshi et al, 1999;Suzuki et al, 2004;Wu et al, 2002). Although SKI is more widely expressed than SnoN in mature hematopoietic cells and play crucial roles in hematopoiesis and myeloproliferative diseases (Pearson-White et al, 1995;Singbrant et al, 2014), very little was known about its roles in differentiation and functions of immune cells. We have previously shown that SKI and SnoN oncoproteins cooperate with phosphorylated STAT3 in an adenocarcinoma lung cancer cell line, HCC827 to repress transcription of the Smad3 gene, which renders the sensitive cells resistant to gefitinib (Makino et al, 2017).…”

t(1;1)(p36;p36) PRDM16/SKI

“…High expression of SKI results in activation of hematopoietic stem cells transcriptional programs and an HSC autonomous repopulating advantage that favors myeloid development through hepatocyte growth factor (HGF) signaling and independently of its ability to inhibit TGFB signaling. SKI is highly expressed in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) (Singbrant et al, 2014). SKI has a critical role in the development of neuronal and muscle cells or tissues.…”

The Hepatocyte Growth Factor (HGF)/Met Axis: A Neglected Target in the Treatment of Chronic Myeloproliferative Neoplasms?

“…The recent report that myeloid progenitors transformed with the proto-oncogene SKI (v-ski or avian sarcoma viral oncogene homolog) later become partially dependent on HGF signalling, also suggests the possible occurrence of independent (passenger) events causing overexpression of HGF, secondary to transformation but not directly dependent from the main oncogenic event [ 134 ]. Altogether, the literature and our own studies support an indirect stimulation of the HGF/Met axis in chronic myeloid malignancies, by other cytokinesorby additional oncogenic events occurredeither in the early stages of transformation or/and during disease progression.…”