Ribonuclease Inhibitor (RNH1) Is a Novel Regulator in Myelopoiesis and Resolves Differentiation Blockade in Acute Myeloid Leukemia
Authors: Saha Dipanjali , Angelillo-Scherrer Anne , Allam Ramanjaneyulu 
Published: December 2024

Introduction:

Ribonuclease Inhibitor (RNH1), conventionally known for it's role in inhibiting pancreatic ribonucleases, has emerged as a regulator of hematopoiesis, with pivotal functions in embryonic development and erythropoiesis. This critical role of RNH1 was highlighted by studies on a mouse model where Rnh1FL/FL mice were backcrossed with Vav1-iCre mice to achieve deletion of Rnh1 specifically in the hematopoietic compartment. In addition to anemic phenotype the Rnh1-deficient mice showed increased myelopoiesis. An interesting observation was that despite the increased myelopoiesis, there were no signs of leukemogenesis in Rnh1-/- mice and the resulting myeloid cells were functional, with the phenotype likened to a subtle form of emergency myelopoiesis.

Acute myeloid leukemia (AML) presents a daunting challenge due to it's heterogeneous nature, characterized by uncontrolled proliferation and differentiation blockage in myeloid progenitors. Despite strides in treatment, hematopoietic stem cell transplantation (HSCT) remains the sole curative therapy and non-HSCT interventions often yield suboptimal outcomes, underscoring the need for enhanced treatment modalities targeting the hallmark obstruction of myeloblast differentiation in AML.

Leveraging these insights, we hypothesized that targeting RNH1 could alleviate myeloid differentiation arrest in AML, offering a novel therapeutic avenue.

Methods: To comprehend the role of RNH1 in myelopoiesis and AML, RNH1-deficient (KO) AML cell lines (THP1, Molm13, HL60 and OCI-AML3) have been generated using the CRISPR-Cas9 method. Subsequent myeloid phenotypic and differentiation experiments were conducted. RNA sequencing, mass spectrometry and downstream biochemical assays and functional assays were conducted to elucidate the underlying mechanisms. Additionally, lentivirus-mediated RNH1 knockdown experiments were performed using peripheral blood and bone marrow (BM) derived mononuclear cells (MNCs) from AML patients. BM cells derived from Rnh1-/- mice were genetically modified in-vitro to introduce AML and then differentiation and proliferating potential of the cells are measured, to further understand how RNH1 manipulates differentiation in context of AML.

Results: In line with RNH1 role in myelopoiesis in mice, loss of RNH1 increases myeloid differentiation in AML cell lines and it is further enhanced upon treatment with differentiation agents, like All-trans retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3. In support of these findings, RNH1 knockdown in blast cells from AML patients increased myeloid differentiation. Transcriptomic analysis in THP1 cells revealed that upon loss of RNH1, certain genes related to myeloid differentiation were upregulated, although the transcription levels of key myeloid transcription factors (TF) such as PU.1 and C/EBP alpha remained unchanged. However, increased protein levels of these TFs was observed, suggesting potential post-transcriptional or translational regulation by RNH1. Most of the essential ribosomal proteins were downregulated leading to a decrease in ribosomal biogenesis in the RNH1 KO cells. RNH1 deletion in other AML cell lines also lead to a defective translation machinery with reduced global translation. Mass spectrometry analysis identified interaction of RNH1 with histone deacetylases (HDAC1 and 2) and the master myeloid TF C/EBPα, indicating possibility of RNH1 mediated epigenetic regulation of myelopoiesis. Preliminary experiments indicate alteration in levels of total and acetylated form of histones between WT and RNH1 KO AML cells. Whether the RNH1 mediated enhancement of myeloid differentiation is attributed to defective ribosomal biogenesis and translation or due to differences in epigenetic regulation of myeloid TFs, is currently under investigation.

Conclusion: Our findings highlight RNH1 as a novel regulator of myeloid differentiation, offering a promising therapeutic target for overcoming the differentiation block in AML. Further elucidation of the molecular mechanisms by which RNH1 mediates myeloid differentiation holds significant potential for advancing AML therapy.