Christina B. Wölwer

Erythroid enucleation is the final stage of erythroid terminal differentiation and involves the separation of an orthochromatic erythroblast into two daughter cells – a pyrenocyte containing the extruded nucleus, and a reticulocyte, which will become a red blood cell. Our previous work has identified CDK9 as a regulator of erythroid enucleation that appears to act independently of its known role in regulating RNA polymerase II transcription, suggesting the potential for a new CDK9 role. Using a co-immunoprecipitation and mass spectrometry approach, we here identified the interactome of CDK9 in differentiating erythroblasts. We show that CDK9 interacts with a RanGTP–importin-β complex during erythroid terminal differentiation, and inhibition of importin-β in erythroblasts blocks erythroid enucleation. Using imaging analysis and functional assays of enucleating erythroblasts, we show that CDK9 and importin-β colocate at a crucial site of activity opposite to the nucleus before nuclear extrusion and we describe a novel finding that physically links CDK9 and importin-β activity prior to calmodulin and Ca2+ signalling, and subsequent F-actin activity, to achieve enucleation.

Erythroid enucleation is the final stage of erythroid terminal differentiation and involves the separation of an orthochromatic erythroblast into two daughter cells; a pyrenocyte containing the extruded nucleus, and a reticulocyte that will become a red blood cell. Our previous work identified CDK9 as a regulator of erythroid enucleation that appears to act independently of its known role in regulating RNA polymerase II transcription, suggesting the potential for a new CDK9 role. Using a co-immunoprecipitation and mass spectrometry approach, we identified the interactome of CDK9 in differentiating erythroblasts. We show that CDK9 interacts with a RanGTP-NEMP1-Importin-β complex during erythroid terminal differentiation, and inhibition of importin-β in erythroblasts blocks erythroid enucleation. Using imaging analysis and functional assays of enucleating erythroblasts, we show that CDK9 and importin-β co-locate at a critical site of activity opposite to the nucleus before nuclear extrusion and we describe a novel finding that physically links CDK9 and importin-β activity prior to CaM/Ca ²⁺ signalling and subsequent F-actin activity to achieve enucleation. Key Points: Importin-β physically interacts with CDK9 in erythroid cells and is a novel regulator of erythroid enucleation CDK9 and importin-β are required upstream of CaM/Ca ²⁺ signalling to enable nuclear extrusion

Introduction The loss of the Y chromosome in various malignant diseases has been described previously. There are no reliable information on the actual frequency, significance and homogeneity of Y chromosome loss (LoY) in esophageal adenocarcinoma (EAC). Aim The study analyzed the relevance of the Y chromosome in EAC. Material and methods 400 male EAC including lymph-node metastases were analyzed with commercially available Y chromosome specific fluorescence in-situ probes. The results were correlated with molecular and immunohistochemical markers and clinicopathological aspects. Results The entire cohort (n=400) showed a singular LoY of one chromosome arm in 1.0% (q-arm) and 2.8% (p-arm), complete LoY in 52.5%. LoY was strongly associated with shortened overall-survival (OS). Patients with preserved Y chromosome had a median OS of 58.8 months, patients with LoY an OS of 19.4 months (p < 0.001). Multivariate analysis showed LoY as an independent prognostic marker with a hazard ratio of 1.835 (95% CI 1.233 - 2.725). LoY correlated with TP53 mutations (p = 0.003), KRAS amplification (p = 0.004), loss of ARID1a (p = 0.045) and presence of LAG3 (p = 0.018). Conclusions Loss of the Y chromosome is a very common phenomenon in EAC. The LoY is heterogeneously distributed within the tumor, but corresponding lymph node metastases frequently show homogeneous LoY, indicating a selection and metastasizing advantage with poor prognosis. Also, an influence on the tumor microenvironment is likely. To date, the male predominance of EAC (7 - 9:1) is unclear, so genetic explanatory models are favored. Our data suggest that LoY has to be seen in a larger functional context. Understanding this context may shed light on the gender bias of EAC incidence. Publication Date: 08 September 2020 (online) © Georg Thieme Verlag KG Stuttgart · New York

Background: The loss of the Y chromosome in various malignant diseases has been described previously. There are no reliable information on the actual frequency, significance and homogeneity of Y chromosome loss (LoY) in esophageal adenocarcinoma (EAC). Methods: 400 male EAC including lymph-node metastases were analyzed with commercially available Y chromosome specific fluorescence in-situ probes. The results were correlated with molecular and immunohistochemical markers and clinicopathological aspects. Results: The entire cohort (n = 400) showed a singular LoY of one chromosome arm in 1.0% (q-arm) and 2.8% (p-arm), complete LoY in 52.5%. LoY was strongly associated with shortened overall-survival (OS). Patients with preserved Y chromosome had a median OS of 58.8 months, patients with LoY an OS of 19.4 months (p < 0.001). Multivariate analysis showed LoY as an independent prognostic marker with a hazard ratio of 1.835 (95% CI 1.233–2.725). LoY correlated with TP53 mutations (p = 0.003), KRAS amplification (p = 0.004), loss of ARID1a (p = 0.045) and presence of LAG3 (p = 0.018). Conclusions: Loss of the Y chromosome is a very common phenomenon in EAC. The LoY is heterogeneously distributed within the tumor, but corresponding lymph node metastases frequently show homogeneous LoY, indicating a selection and metastasizing advantage with poor prognosis. To date, the male predominance of EAC (7–9:1) is unclear, so genetic explanatory models are favored. The LoY in EAC may be biologically and functionally relevant and additional genomic or functional analyses are needed.