RUNX1 Mutations Cause a Myeloid Differentiation Block Leading to the Formation of a Long Term Expanding CD34+/CD33+/CD45RA+/CD123+ Cell Population

RUNX1 (AML1) is a transcription factor critically involved in normal haematopoiesis. Inactivating RUNX1 mutations have been frequently described in a variety of myeloid neoplasms, including high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Here, we aimed to functionally and molecularly define the actions of a dominant negative mutant by in vitro and in vivo experiments and RNA- and ChIP-sequencing approaches. Overexpression of the RUNX1 mutant S291fs300X in cord blood (CB) CD34+ cells caused a decline in erythroid colony formation (p= 0.01) while the CFU-GM colonies showed enhanced replating capacity compared to control (>3 times). It appeared that the replating potential was restricted to CD14-/CD15- progenitor cells. Long-term suspension cultures with myeloid growth factors (IL-3, SCF) of RUNX1 S291fs300X CB CD34+ cells provided a rather homogenous cell population after 10 weeks of culture. These cells are growth factor dependent and are phenotypically defined by CD34+/CD38+/CD33+/IL1-RAP+/CD45RA+/CD123+ resembling a GMP phenotype which can be propagated for approximately 20 weeks in suspension. Comparable results were obtained with normal bone marrow CD34+cells transduced with the RUNX1 S291fs300X. Karyotype analyses demonstrated no abnormalities while integration site analysis showed a variety of different integration sites and differences between individual samples, suggesting that the myeloid differentiation block is related to the RUNX1 S291fs300X mutation.Long-term MS5 stromal co-cultures of transduced RUNX1 S291fs300X CB CD34+ cells showed after 8-10 weeks a rather homogenous cell population with limited potential to expand and localized under the stromal layer. This cell population is phenotypically defined by CD34+/CD38-. The interactions with the stroma appear to prevent proliferation but retain quiescence, indicating that sufficient niche-cell interactions might be crucial for transformation. NSG mice experiments are performed to test the reproducibility of these findings in vivo. Q-PCR studies demonstrated reduced expression of C/EBPα in RUNX1 S291fs300X CB CD34+ cells, one of the key targets in myeloid differentiation. Therefore, week 10 RUNX1 S291fs300X CB CD34+ cells were transduced with a retroviral C/EBPα overexpression vector. The re-expression of C/EBPα resulted in a reduction in cell proliferation, decline of undifferentiated blasts and an increase in CD15 expression. RNA- and ChIP-sequencing data revealed a decreased expression of crucial RUNX1 target genes including C/EBPα and Cited2 and also a retained binding of mutant RUNX1 on these loci in conjunction with a decrease of H3K27ac. Further research into the molecular mechanisms by which this RUNX1 S291fs300Xderegulates gene-expression is in progress. Our results implicate that overexpression of RUNX1 S291fs300X mutant leads to impaired erythroid differentiation and a strong differentiation block of the myeloid lineage resulting in the expansion and maintenance of a GMP-like cell population. Disclosures No relevant conflicts of interest to declare.

Primary Dural Melanoma

This study reviews the literature pertaining to primary meningeal melanoma and reports the clinical and ultrastructural findings in a case where the tumor appeared to be of pachymeningeal (dural) origin. This is clearly a departure from all previously described cases, in which a leptomeningeal (pial-arachnoidal) origin was either defined or assumed. Clinically. this case was remarkable in its rarity. its presentation as a cerebellopontine angle syndrome, and its occurrence in a Negro. a race in which melanomas are uncommon. Ultrastructurally. the tumor did demonstrate the presence of basement membrane abnormalities and numerous endothelial fenestrations. However, it was found to be made up of a homogenous cell population, consisting only of electron-lucent, melanin-laden cells. The mixed cell population noted previously in a primary leptomeningeal melanoma was not found in this tumor. In view of the fact that this patient continues to do well 1/12; years after operation, with no evidence of tumor recurrence, it is suggested that a homogenous cell population noted on electron microscopy could indicate a better prognosis. In addition, it may also indicate a pachymeningeal rather than a leptomeningeal origin for the tumor. A plea is made for greater specificity in terminology when describing primary meningeal melanomas and for a concerted effort to distinguish between those of dural and those of leptomeningeal origin.

Proliferation Kinetics of Acute Leukemia Cells in Relation to the Chemotherapy

SummaryThe proliferative characteristics of human acute leukaemia cells are reported and the relationships between the proliferative alterations and differentiation defect of these cells discussed. The proliferative activity of acute leukaemia cells was also studied in relation to cytostatic treatment.Emphasis is laid on the fact that in all cases of acute leukaemia the characteristic blast cells of the disease do not constitute a homogenous cell population but can be divided into various sub-classes with different kinetic and proliferative characteristics. It is also pointed out that all cytostatic treatment acts on the most actively proliferating classes and only indirectly on the non-proliferating classes.Finally, the need for more detailed study of DNA synthesis at chromosome and sub-chromosome level for the purpose of more fully understanding the response of leukaemic cells to the various chemiotherapies is underlined.