Transcription factor 3 is dysregulated in megakaryocytes in myelofibrosis

Abstract

Transcription factor 3 (TCF3) is a DNA transcription factor that modulates megakaryocyte development. Although abnormal TCF3 expression has been identified in a range of hematological malignancies, to date, it has not been investigated in myelofibrosis (MF). MF is a Philadelphia-negative myeloproliferative neoplasm (MPN) that can arise de novo or progress from essential thrombocythemia [ET] and polycythemia vera [PV] and where dysfunctional megakaryocytes have a role in driving the fibrotic progression. We aimed to examine whether TCF3 is dysregulated in megakaryocytes in MPN, and specifically in MF. We first assessed TCF3 protein expression in megakaryocytes using an immunohistochemical approach analyses and showed that TCF3 was reduced in MF compared with ET and PV. Further, the TCF3-negative megakaryocytes were primarily located near trabecular bone and had the typical “MF-like” morphology as described by the WHO. Genomic analysis of isolated megakaryocytes showed three mutations, all predicted to result in a loss of function, in patients with MF; none were seen in megakaryocytes isolated from ET or PV marrow samples. We then progressed to transcriptomic sequencing of platelets which showed loss of TCF3 in MF. These proteomic, genomic and transcriptomic analyses appear to indicate that TCF3 is downregulated in megakaryocytes in MF. This infers aberrations in megakaryopoiesis occur in this progressive phase of MPN. Further exploration of this pathway could provide insights into TCF3 and the evolution of fibrosis and potentially lead to new preventative therapeutic targets.

Plain Language Summary

What is the context?

• We investigated TCF3 (transcription factor 3), a gene that regulates megakaryocyte development, for genomic and proteomic changes in myelofibrosis.

• Myelofibrosis is the aggressive phase of a group of blood cancers called myeloproliferative neoplasms, and abnormalities in development and maturation of megakaryocytes is thought to drive the development of myelofibrosis.

What is new?

• We report detection of three novel TCF3 mutations in megakaryocytes and decreases in TCF3 protein and gene expression in primary megakaryocytes and platelets from patients with myelofibrosis.

• This is the first association between loss of TCF3 in megakaryocytes from patients and myelofibrosis.

What is the impact?

• TCF3 dysregulation may be a novel mechanism that is responsible for the development of myelofibrosis and better understanding of this pathway could identify new drug targets.

Keywords:

• Megakaryocyte
• myelofibrosis
• myeloproliferative neoplasms
• next-generation sequencing
• platelets
• TCF3

Acknowledgments

We acknowledge Alan Morling, Brett Bettridge and Aaron Osborn from PathWest, Royal Perth Haematology, Sir Charles Gairdner Hospital Haematology and Rockingham General Haematology for their assistance with sample collection.

Author contributions

R.J.C., B.B.G. and M.D.L. designed and performed the research. R.J.C. and B.B.G. analyzed the data. B.M., L.W. and D.B. isolated platelets. B.B.G, B.M., L.W. and K.A.F. isolated megakaryocytes. ZY.N., H.C., R.H., C.S.G., J.A.J.M., and M.F.L. recruited patients and analyzed the clinical data. H.C., R.J.C., W.N.E. and ZY.N. reviewed and analyzed the bone marrow trephines. R.J.C. and W.N.E reviewed the immunohistochemistry-stained sections. R.J.C. performed the pathway analysis. All authors contributed to drafting the paper and had final approval of the submitted and published versions.

Disclosure statement

The authors declare that they have no competing interests.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Additional information

Funding

The work was supported by grants from the Cancer Council Western Australia (APP1065493 and CCWA ECCRoY 2016), the University of Western Australia (RA/1/1200/883), the MPN Research Foundation and the Ruby Red Foundation (RA/1/3246/2 and RA/1/3472/2), Ann Helene Toakley Charitable Endowment (IPAP2018/1802, IPAP2019/0291) and Raine Medical Research Foundation (RPG043-19). R.J.C. is supported by Research Training Program Scholarship provided by the University of Western Australia. B.B.G. is supported by the Gunn Family National Career Development Fellowship for Women in Haematology from Snowdome Foundation and Maddie Riewoldt’s Vision. M.D.L. was an ISAC Marylou Ingram Scholar. ZY.N is supported by a Fellowship of the Western Australia Cancer and Palliative Care Network Health from the Western Australia Health Department. H.C. was supported by a Fellowship of the Western Australia Cancer and Palliative Care Network Health from the Western Australia Health Department. J.A.J.M. received financial support from the Royal College of Pathologists of Australasia.