No abstract

Adult stem and progenitor cells are uniquely capable of self-renewal, and targeting this process represents a potential therapeutic opportunity. The early erythroid progenitor, burst-forming unit erythroid (BFU-E), has substantial self-renewal potential and serves as a key cell type for the treatment of anemias. However, our understanding of mechanisms underlying BFU-E self-renewal is extremely limited. Here, we found that the muscarinic acetylcholine receptor, cholinergic receptor, muscarinic 4 (CHRM4), pathway regulates BFU-E self-renewal and that pharmacological inhibition of CHRM4 corrects anemias of myelodysplastic syndrome (MDS), aging, and hemolysis. Genetic down-regulation of CHRM4 or pharmacologic inhibition of CHRM4 using the selective antagonist PD102807 promoted BFU-E self-renewal, whereas deletion ofChrm4increased erythroid cell production under stress conditions in vivo. Moreover, muscarinic acetylcholine receptor antagonists corrected anemias in mouse models of MDS, aging, and hemolysis in vivo, extending the survival of mice with MDS relative to that of controls. The effects of muscarinic receptor antagonism on promoting expansion of BFU-Es were mediated by cyclic AMP induction of the transcription factor CREB, whose targets up-regulated key regulators of BFU-E self-renewal. On the basis of these data, we propose a model of hematopoietic progenitor self-renewal through a cholinergic-mediated “hematopoietic reflex” and identify muscarinic acetylcholine receptor antagonists as potential therapies for anemias associated with MDS, aging, and hemolysis.

B‐cell novel protein‐1 (BCNP1) or Family member of 129C (FAM129C) was identified as a B‐cell‐specific plasma‐membrane protein. Bioinformatics analysis predicted that BCNP1 might be heavily phosphorylated. The BCNP1 protein contains a pleckstrin homology (PH) domain, two proline‐rich (PR) regions and a Leucine Zipper (LZ) domain suggesting that it may be involved in protein‐protein interactions. Using The Cancer Genome Atlas (TCGA) data sets, we investigated the correlation of alteration of the BCNP1 copy‐number changes and mutations in several cancer types. We also investigated the function of BCNP1 in cellular signalling pathways. We found that BCNP1 is highly altered in some types of cancers and that BCNP1 copy‐number changes and mutations co‐occur with other molecular alteration events for TP53 (tumour protein P53), PIK3CA (Phosphatidylinositol‐4,5‐Bisphosphate 3‐Kinase, Catalytic Subunit Alpha), MAPK1 (mitogen‐activated protein kinase‐1; ERK: extracellular signal regulated kinase), KRAS (Kirsten rat sarcoma viral oncogene homolog) and AKT2 (V‐Akt Murine Thymoma Viral Oncogene Homolog 2). We also found that PI3K (Phoshoinositide 3‐kinase) inhibition and p38 MAPK (p38 mitogen‐activated protein kinase) activation leads to reduction in phosphorylation of BCNP1 at serine residues, suggesting that BCNP1 phosphorylation is PI3K and p38MAPK dependent and that it might be involved in cancer. Its degradation depends on a proteasome‐mediated pathway.

No abstract

Patients with MPNs share common characteristics including overproduction of myeloid cells and chronic inflammation. In MPNs, bromodomain and extra-terminal (BET) proteins can bind to acetylated chromatin to regulate the activity of transcription factors such as NF-kB, leading to overproduction of inflammatory cytokines. Dysregulation of transcriptional gene expression can drive disease defining characteristics, therefore, targeting BET proteins could be an attractive therapeutic approach. In preclinical models, BET inhibitors decreased inflammatory cytokine production and restored hematopoietic cell differentiation. Combination of BET inhibitors with the JAK1/2 inhibitor, ruxolitinib, resulted in further reduction in cytokine production and bone marrow fibrosis (Kleppe M. Cancer Cell 2018). Here, we investigate a novel, orally bioavailable BET inhibitor, INCB057643, for the treatment of MPN in combination with ruxolitinib. INCB057643 is a potent and selective inhibitor of the BET family. In binding assays, INCB057643 selectively inhibited both BRD4 bromodomains, BD1 and BD2, with IC50 values of 39 nM and 6 nM, respectively. INCB057643 comparably inhibited both NF-kB reporter activity and MYC protein levels. Nascent transcript analysis on the JAK2 V617F-mutant cell line SET2 showed decreased transcription of genes linked to inflammation, alone and in combination with ruxolitinib. These data suggest that INCB057643 and ruxolitinib can be used in combination to effectively suppress the pathogenic gene program driving chronic inflammation. Functionally, INCB057643 inhibited proliferation of SET2 cells at nanomolar concentrations. In a SET2 xenograft model, combination of INCB057643 and ruxolitinib potently inhibited tumor growth. Similarly, in the MPLW515L-driven MPN mouse model, INCB057643 in combination with ruxolitinib resulted in a greater reduction in spleen volume, compared to that achieved with single agent treatment. Meso scale discovery assays on MPN-patient-derived CD34+ cells and whole blood using INCB057643 showed inhibition of NF-kB-mediated production of cytokines such as IL-8. In addition, flow cytometry-based analysis on MPN-patient-derived CD34+ cells showed that INCB057643 in combination with ruxolitinib decreased pathogenic megakaryopoiesis, and increased terminal erythroid differentiation. Our data demonstrate that the combination of the BET inhibitor, INCB057643, with ruxolitinib can restore normal hematopoietic differentiation, reduce inflammatory gene expression, and inhibit pathogenic cell differentiation in preclinical models of MPN. INCB057643 is currently being evaluated in clinical studies as monotherapy and in combination with ruxolitinib in patients with MPN. Acknowledgments: Michelle Pusey and Gaurang Trivedi contributed equally to this study. Citation Format: Michelle Pusey, Gaurang Trivedi, Bob Collins, Nina Zolotarjova, Pat Feldman, Monica Bomber, Jacob Ellis, Kristy Stengel, Cynthia Timmers, Hamza Celik, Matt Stubbs, Jeff Jackson, Scott Hiebert, Susan Wee, Sunkyu Kim. Preclinical characterization of the BET inhibitor, INCB057643, in combination with ruxolitinib for treatment of myeloproliferative neoplasms (MPN). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6274.