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D. Ingenhag et al.
human HT1080 and murine NIH3T3 cells. In both cell-line models HB9-expression led to growth arrest within 72 h after transduction, mediated via the p53-p21 signaling axis. In line with this, siRNA-mediated p53-knockdown abolished the HB9-dependent growth inhibitory effect. Regarding cell cycle analysis, HT1080[HB9] showed a sig- nificant decrease in cells at the S-phase, stalling the cells at G1/G2-phase of the cell cycle. NIH3T3[HB9] corroborated that effect, and in addition exhibited a decrease in cells in the G1-phase and an increase in aneuploid cells, which can be explained as a dose-dependent effect, as NIH3T3 showed lower HB9 expression levels upon transduction compared to HT1080. This leads to less activation of p53- signaling, resulting in an incomplete G1/G2 arrest, so that NIH3T3[HB9] cells have the chance to re-enter the cell cycle, irrespective of an inappropriate replication, leading to an increase of aneuploid cells (> 4n), while there is a decrease in diploid cells at G1- and S-phase. Besides onset of a tumor-suppressor network, resulting in growth arrest, HB9-expressing cells exhibited typical morphological changes, becoming flattened, enlarged and multinuclear, as well as expression of SA-β-gal, thus fulfilling all main criteria of senescence.1
Cellular senescence is initiated in response to replicative stress, resulting from irreversible DNA damage, and can be differentiated into replicative senescence and prema- ture senescence. Induction of premature senescence has been shown for many potent oncogenes, such as RAS, RAF, MEK and BRAF.4,36-38 In contrast to replicative senes- cence, oncogene-induced premature senescence occurs independently of telomer attrition, as a result of a strong mitogenic signal, which leads to an inappropriate replica- tion of the DNA during the S-phase of the cell cycle and thus activation of DNA damage response.2 In line with this, HB9 triggered a DNA damage response in HT1080 and NIH3T3, indicated by phosphorylation of p53 at Ser15, as this specific phosphorylation site is a target for the DNA-damage kinases ATM and ATR.31,39 Thus HB9 expression induces DNA damage, which in turn activates p53-signaling as part of the DNA damage response. Furthermore, HB9-dependent replicative stress led to an increase in aneuploid cells in NIH3T3[HB9], which has already been shown for MYC oncogene.3 This was further validated in HB9-transduced CD34+ HSPCs: RNA-Seq analyses revealed a positive enrichment of biological processes related to DNA/RNA processing, as well as cell cycle and mitosis, and a negative enrichment of processes related to post-translational phosphorylation and intracel- lular signaling (Online Supplementary Table S2 and Online Supplementary Figure S16), which correlates to the replica- tive stress-dependent expression profile of MYC-driven lymphoma.40
Oncogenes, which induce premature senescence in vitro, are known to cause pre-malignant cell populations in vivo.35 This was shown for murine lung adenomas (RASV12), T-cell lymphomas (RASV12), prostate tumors (PTEN), as well as human benign melanocytic naevi (BRAFE600).38,41-43
With respect to translocation t(7;12) leukemogenesis, we set up a murine bone marrow transplantation model to investigate whether ectopic HB9-expression affects hematopoietic cell differentiation in vivo. Therefore, murine HSPCs were transduced with either HB9-GFP or GFP vector and transplanted into myeloablative irradiated recipient mice. HB9-transduction of Lin– HSPCs yielded
expression levels comparable to that of translocation t(7;12) AML blast cells.22 Similar to human HSPCs, no endogenous HB9-expression was detectable in murine Lin– HSPCs.15,20,21 In vivo, HB9-expressing HSPCs under- went an overall differentiation arrest, as no HB9-GFP+ cells were detected within the mature B-, T- and myeloid-cell pool of recipient mice. Analysis of the HSPC compart- ment revealed an early blockage of the lymphoid lineage, while HB9-expressing HSPCs showed a strong myeloid lineage commitment. Within the myeloid progenitor pop- ulation HB9-expressing cells were significantly enriched in the MEP, compared to the CMP and GMP compartment, thus revealing proliferation of HB9+ cells arrested at the MEP stage. Instead a differentiation blockage without pro- liferation would have resulted in comparable frequencies of GMP and MEP, whereas a megakaryocytic- and/or ery- throid-biased differentiation would have resulted in com- parable frequencies of CMP and MEP.
A myeloid-biased differentiation in combination with a diminished potential of lymphoid differentiation is associ- ated with an aged hematopoietic system. This is assumed to result from aged HSCs, which show an impaired lym- phoid differentiation capacity, while the myeloid differen- tiation capacity is maintained or even increased.44,45 Consistently, the incidence of myeloid malignancies increases with age. Recent studies highlight not only a myeloid, but in particular a megakaryocytic/erythroid bias in aged human and murine HSCs46 directly correlating to our results of a megakaryocytic/erythroid-biased differen- tiation and de novo expression of erythropoiesis-related genes in HB9+ HSPCs. Furthermore, the in vivo experi- ments have shown that the impaired differentiation capacity of HB9+ HSCs results in a decreased bone mar- row and peripheral blood cellularity throughout the entire monitoring period. This is in line with our findings of HB9-dependent reduced clonogenicity in human CD34+ HSPCs, and further strengthens the assumption that HB9 induces senescence in hematopoietic cells. Onset of HB9- dependent senescence in hematopoietic cells is further supported by RNA-Seq data of CD34+ HSPCs. A strong positive enrichment of mitosis-related processes, together with a contradictory negative enrichment of cytoskeleton organization-related processes, reflects the senescence- associated multinuclear phenotype observed in the cell- line models, at molecular basis in CD34+ HSPCs.
With regard to translocation t(7;12) AML, our data may suggest that HB9-expression dictates the development of exclusively myeloid leukemia in translocation t(7;12)-pos- itive AML patients.15 Although most translocation t(7;12) AML blast cells show a very undifferentiated state, some are defined as erythroblastic17,47 as well as megakaryoblas- tic leukemia,48,49 correlating to the megakaryocytic/ery- throid-biased differentiation due to HB9 expression.
With regard to leukemogenesis, secondary genetic alter- ations are necessary, as sole HB9-expression did not result in complete transformation and progression to AML.
Based on our in vitro results, HB9 expression led to an increase in aneuploid cells, which is correlated to genetic instability. Thus, via induction of genetic instability, HB9 may increase the chance for secondary genetic alterations, which are necessary for complete cellular transformation. Initial screening studies using a panel of frequently mutat- ed genes in AML (NPM1, CEPBA, MLL, WT1, FLT3, N- RAS, K-RAS, PTPN11 and KIT) did not succeed in identi- fying secondary recurrent genetic alterations in transloca-
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haematologica | 2019; 104(1)