Abstract:
The underlying study is based on the research work published in Blood by Skokowa et al. in 2014. The authors postulated a unique mechanism of leukemogenesis in a group of patients suffering from congenital neutropenia (CN), a disease characterized by a low absolute neutrophil count and a high susceptibility of malignant progression to MDS or AML, which occurs in approximately 20 % of CN patients [Skokowa et al., 2014, 2017]. They found that, in approximately 70 % of CN/AML patients, in addition to the inherited CN-associated mutations (e.g., ELANE, HAX1, GPT1 and WAS), the AML phenotype was observed when hematopoietic cell clones were positive for sporadic RUNX1 mutations which were acquired after sporadic CSF3R mutations. The authors postulated cooperating leukemogenic effects of RUNX1 and CSF3R mutations.
In this Doctoral Thesis, we re-analyzed the CN/AML patients’ group investigated in 2014. Among those patients who underwent malignant transformation, we found hints of non-random distribution for sporadic missense (n = 7) and nonsense (n = 6) RUNX1 mutations (Fisher’s t-test: p = 0.0515) at AML stage. Furthermore, samples positive for missense RUNX1 mutations were also positive for trisomy 21. This was not observed in samples positive for nonsense RUNX1 mutations (table 3.1).
Since RUNX1 is located on chromosome 21, it was of special interest to test whether trisomy 21 resulted in an increase of the mutant or the wild-type RUNX1 allelic fraction. Thus, we performed Sanger sequencing and digital PCR on samples of three selected CN/AML individuals all positive for missense RUNX1 mutations and trisomy 21 (UniProtKB:Q01196, p.R139G, p.D171N, p.R174L) (figures 3.1 to 3.6). We were able to confirm an increase of mutant RUNX1 allelic fraction over wild type RUNX1 allelic fraction in a 2:1 ratio in all three patients. Hence, we showed that the occurrence of trisomy 21 was accompanied by an increase of the mutant RUNX1 allele. Since in our patient cohort nonsense RUNX1 mutations were not associated with trisomy 21, we concluded that this was due to different mechanisms of leukemogenic progression between both groups (figure 4.1). Furthermore, we established a chromatin immunoprecipitation assay using a RUNX1 antibody which allows the identification of binding patterns of different mutated RUNX1 proteins to DNA or to other proteins, interaction partners of RUNX1 protein in the future. This might contribute to the better understanding of the patho-mechanisms underlying the effects of different RUNX1 mutations in leukemogenesis.
The second objective reported in this thesis, was to investigate the role of microRNAs in CN pathogenesis. MicroRNAs are small, approximately 22 nts long noncoding RNAs, which exert diverse biologic functions including the post- transcriptional control of mRNAs [Lee, 1993]. First, we established a workflow for the isolation and expression quantification of microRNAs in CN patients. We were able to isolate and quantify microRNA-125b and let-7b and aimed to investigate microRNA-3151 (figures 3.11 to 3.14). We observed that miR-125b expression levels were down-regulated upon myeloid differentiation from CD34+ hematopoietic stem and progenitor cells to CD33+ promyelocytic cells. Those findings were in line with previous reports [O’Connell et al., 2011; Rajasekhar et al., 2018; Shaham et al., 2012]. Interestingly, we could not detect significant differences in miR- 125b expression levels between healthy donors and CN patients, neither in CD34+ nor in CD33+ cell populations. This was also true for miR-125b expression lev- els in CN samples, when grouped according to their inherited mutations (ELANE, HAX1, etc.). Of note, miR-3151 expression was not detected in any of the samples; either because it is not expressed by the cells investigated or due to technical issues of the methods used. In this study, we successfully identified and quantified microRNAs, known to be relevant for hematopoiesis, for the first time in our patient cohort. However, due to the small sample size and the small number of microRNAs examined, further research in this field is required in order to finally draw significant conclusions about the role of microRNA in CN pathogenesis.
In summary, this study expands the understanding of leukemogenic progression in CN and provides valuable workflows for further investigation of the role of RUNX1 proteins as well as microRNA profiles in CN.
