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2531 Ribonuclease Inhibitor 1 (RNH1) Regulates Myeloid Lineage Choice through Cyclin-Dependent Kinase 1

Program: Oral and Poster Abstracts
Session: 501. Hematopoietic Stem and Progenitor Cells and Hematopoiesis: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Fundamental Science, Research, Acute Myeloid Malignancies, AML, adult, Translational Research, hematopoiesis, Diseases, Myeloid Malignancies, Biological Processes, Human, Study Population, Animal model
Sunday, December 11, 2022, 6:00 PM-8:00 PM

Nicola Daniele Andina, MD, PhD1*, Mayuresh Anant Sarangdhar1,2,3*, Aubry Tardivel, MSc4*, Camille Ansermet, PhD5*, Giuseppe Bombaci, PhD1,6*, Mahmoud Hallal, PhD1,6,7*, Vijay Kumar Chennupati, PhD8*, Yara Banz, MD, PhD9*, Manfred Heller, PhD1*, Anne Angelillo-Scherrer, MD-PhD1,2,10, Nicolas Bonadies, MD11,12 and Ramanjaneyulu Allam, PhD1,2*

1Department for BioMedical Research, University of Bern, Bern, Switzerland
2Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, Bern, Switzerland
3Department of Hematology and Central Laboratory of Hematology, University Bern, Bern, Switzerland
4University Bern, Bern, CHE
5DBMR, University Bern, Bern, Switzerland
6Department of Hematology and Central Laboratory of Hematology, Inselspital, Bern University Hospital, Bern, Switzerland
7Department of Haematology and Central Haematology Laboratory, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland
8Ludwig Center for Cancer Research, University of Lausanne, Lausanne, Switzerland
9Institute of Pathology, University of Bern, Bern, Switzerland
10Inselspital, Department of Hematology and Central Hematology Laboratory, University Hospital Bern, Bern, Switzerland
11Department of Hematology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland
12Department of BioMedical Research, University of Bern, Bern, Switzerland

Background: Myelopoiesis is a complex process, it begins with myeloid lineage choice determination in haemopoietic stem cells (HSCs). Still we do not have complete molecular understanding of how myeloid lineage choices were determined. Although transcription factors expression majorly determine the lineage choices at HSCs level, recent study suggest that ratios of total PU.1:GATA1 protein levels are not the central mechanism that initiates HSPC lineage decisions . Further, cell fate commitment is linked to the cell cycle regulation, however whether the cell cycle itself regulates lineage choices in HSCs is not known. Previously, we have published that Ribonuclease inhibitor (RNH1) deletion in mice leads to defect inembryonic erythropoiesis. Here, we report that RNH1 regulates myeloid lineage choice through cyclin-dependent kinase 1 (CDK1) mediated cell cycle regulation.

Methods: We have generated hematopoietic-specific conditional knockout mice by crossing Rnh1FL/FL mice with Vav1-iCre and Mx1-Cre mice, respectively. Phenotypic analysis, Bone marrow transplantation (BMT) experiments, cell cycle and CFU assays were performed. To understand molecular mechanism RNA seq, mass spectrometry, confocal microscopy and biochemical studies were performed. Histopathology performed in AML bone marrow biopsies. RNH1 was knocked out in several AML cell lines and analyzed myeloid differentiation and performed CFU assays.

Results: Rnh1-deficiency in both Vav1-iCre and Mx1-Cre models resulted in elevated levels of myeloid cells but decreased lymphoid cells and haemoglobin (HGB) levels in peripheral blood. Total bone-marrow (BM) cellularity was similar in wild type (WT) and Rnh1-/- mice. However, the number of erythroid cells and lymphoid cells was significantly decreased, whereas myeloid cells were significantly increased. We analysed the progenitor populations in the BM. In line with increased myelopoiesis phenotype, granulocyte-monocyte progenitor (GMP) cell numbers were increased but common lymphoid progenitor (CLP) and megakaryocyte-erythrocyte progenitor (MEP) cell numbers were decreased. Interestingly, we found increased numbers of long term HSCs (LT-HSCs) and short term HSCs (ST-HSCs) in Rnh1-/- mouse BM, suggesting that RNH1 could affect HSC function. Supporting this, Rnh1-/- HSCs failed to engraft lethally irradiated mice in competitive and non- competitive BMT experiments.

Intriguingly, despite increased myelopoiesis in Rnh1-/- mice, conventional myeloid genes that support myelopoiesis were not increased in Rnh1-/- HSCs by RNA-Seq studies. Rather we found increased expression of genes related to cell cycle, kinetochore and DNA damage in Rnh1-/- HSCs. Corroborating this, Rnh1-/- HSCs showed significant increase in G1 cycle phase compared to WT. At the molecular level, we found that RNH1 directly binds to CDK1 and also inhibits CDK1 kinase function. Supporting this, molecular docking studies suggest that RNH1 potentially binds with CDK1. In agreement with this, loss of RNH1 in HSCs increased CDK1 protein levels as well as increased CDK1 phosphorylation at Thr161. Further, CDK1 inhibitor treatment decreased myelopoiesis and rescued erythropoiesis and lymphopoiesis in Rnh1-/- mice.

We checked whether RNH1 mediated myelopoiesis is relevant in any disease models and can be targeted. Interestingly, we found increased RNH1 expression in AML patients bone marrow biopsies independent of their genetic mutation. AML blasts have myeloid differentiation arrest and therapies that resolve differentiation arrest represent a powerful treatment strategy. Interestingly, RNH1 deletion in AML cell lines leads to increased expression of the myeloid markers, increased cell death and formed less colonies compared to wild type cells.

Summary: Collectively, our studies suggest that loss of RNH1 in HSCs leads to dysregulation in cell cycle through increased CDK1 activity. Dysregulation in cell cycle decreases HSCs stemness and repopulating potential, and influences cell fate decisions by increasing myelopoiesis. Since loss of RNH1 increased myelopoiesis without increase of myeloid transcription factors expression and dependent on CDK1, we call this pathway 'alternative myelopoiesis'. Targeting alternative myelopoiesis could be a potential therapy for AML.

Disclosures: Angelillo-Scherrer: SNSF: Research Funding; Vaderis: Consultancy; Pfizer: Other: Speaker fees; Silence Therapeutics: Research Funding.

*signifies non-member of ASH