Epidermal growth factor receptor-dependent DNA repair promotes murine and human hematopoietic regeneration.

Chemotherapy and irradiation cause DNA damage to hematopoietic stem cells (HSCs), leading to HSC depletion and dysfunction and the risk of malignant transformation over time. Extrinsic regulation of HSC DNA repair is not well understood, and therapies to augment HSC DNA repair following myelosuppression remain undeveloped. We report that epidermal growth factor receptor (EGFR) regulates DNA repair in HSCs following irradiation via activation of the DNA-dependent protein kinase-catalytic subunit (DNA-PKcs) and nonhomologous end joining (NHEJ). We show that hematopoietic regeneration in vivo following total body irradiation is dependent upon EGFR-mediated repair of DNA damage via activation of DNA-PKcs. Conditional deletion of EGFR in hematopoietic stem and progenitor cells (HSPCs) significantly decreased DNA-PKcs activity following irradiation, causing increased HSC DNA damage and depressed HSC recovery over time. Systemic administration of epidermal growth factor (EGF) promoted HSC DNA repair and rapid hematologic recovery in chemotherapy-treated mice and had no effect on acute myeloid leukemia growth in vivo. Further, EGF treatment drove the recovery of human HSCs capable of multilineage in vivo repopulation following radiation injury. Whole-genome sequencing analysis revealed no increase in coding region mutations in HSPCs from EGF-treated mice, but increased intergenic copy number variant mutations were detected. These studies demonstrate that EGF promotes HSC DNA repair and hematopoietic regeneration in vivo via augmentation of NHEJ. EGF has therapeutic potential to promote human hematopoietic regeneration, and further studies are warranted to assess long-term hematopoietic effects.

Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution.

Ionizing radiation and chemotherapy deplete hematopoietic stem cells and damage the vascular niche wherein hematopoietic stem cells reside. Hematopoietic stem cell regeneration requires signaling from an intact bone marrow (BM) vascular niche, but the mechanisms that control BM vascular niche regeneration are poorly understood. We report that BM vascular endothelial cells secrete semaphorin 3 A (SEMA3A) in response to myeloablation and SEMA3A induces p53 - mediated apoptosis in BM endothelial cells via signaling through its receptor, Neuropilin 1 (NRP1), and activation of cyclin dependent kinase 5. Endothelial cell - specific deletion of Nrp1 or Sema3a or administration of anti-NRP1 antibody suppresses BM endothelial cell apoptosis, accelerates BM vascular regeneration and concordantly drives hematopoietic reconstitution in irradiated mice. In response to NRP1 inhibition, BM endothelial cells increase expression and secretion of the Wnt signal amplifying protein, R spondin 2. Systemic administration of anti - R spondin 2 blocks HSC regeneration and hematopoietic reconstitution which otherwise occurrs in response to NRP1 inhibition. SEMA3A - NRP1 signaling promotes BM vascular regression following myelosuppression and therapeutic blockade of SEMA3A - NRP1 signaling in BM endothelial cells accelerates vascular and hematopoietic regeneration in vivo.

E47 Governs the MYC-CDKN1B/p27KIP1-RB Network to Growth Arrest PDA Cells Independent of CDKN2A/p16INK4A and Wild-Type p53.

BACKGROUND & AIMS: Oncogenic mutations in KRAS, coupled with inactivation of p53, CDKN2A/p16INK4A, and SMAD4, drive progression of pancreatic ductal adenocarcinoma (PDA). Overexpression of MYC and deregulation of retinoblastoma (RB) further promote cell proliferation and make identifying a means to therapeutically alter cell-cycle control pathways in PDA a significant challenge. We previously showed that the basic helix-loop-helix transcription factor E47 induced stable growth arrest in PDA cells in vitro and in vivo. Here, we identified molecular mechanisms that underlie E47-induced growth arrest in low-passage, patient-derived primary and established PDA cell lines. METHODS: RNA sequencing was used to profile E47-dependent transcriptomes in 5 PDA cell lines. Gene Ontology analysis identified cell-cycle control as the most altered pathway. Small interfering RNA/short hairpin RNA knockdown, small-molecule inhibitors, and viral expression were used to examine the function of E47-dependent genes in cell-cycle arrest. Cell morphology, expression of molecular markers, and senescence-associated ß-galactosidase activity assays identified cellular senescence. RESULTS: E47 uniformly inhibited PDA cell-cycle progression by decreasing expression of MYC, increasing the level of CDKN1B/p27KIP1, and restoring RB tumor-suppressor function. The molecular mechanisms by which E47 elicited these changes included altering both RNA transcript levels and protein stability of MYC and CDKN1B/p27KIP1. At the cellular level, E47 elicited a senescence-like phenotype characterized by increased senescence-associated ß-galactosidase activity and altered expression of senescence markers. CONCLUSIONS: E47 governs a highly conserved network of cell-cycle control genes, including MYC, CDKN1B/p27KIP1, and RB, which can induce a senescence-like program in PDA cells that lack CDKN2A/p16INK4A and wild-type p53. RNA sequencing data are available at the National Center for Biotechnology Information GEO at https://www.ncbi.nlm.nih.gov/geo/; accession number: GSE100327.

A screen for inducers of bHLH activity identifies pitavastatin as a regulator of p21, Rb phosphorylation and E2F target gene expression in pancreatic cancer.

The average survival for patients with Pancreatic Ductal Adenocarcinoma (PDA) is merely 6 months, underscoring the need for new therapeutic approaches. During PDA progression, pancreatic acinar cells lose activity of the ClassI/II bHLH factors that regulate quiescence. We previously found that promoting transcriptional activity of the Class I bHLH factor E47 in highly aggressive PDA cells induced stable growth arrest in vitro and in vivo. To translate these findings for clinical utility, we developed a high throughput screening platform to identify small molecule inducers of Class I/II bHLH activity. A screen of 4,375 known drugs identified 70 bHLH activators. Prominent among the hits were members of the statin class of HMG-CoA reductase inhibitors, cholesterol lowering drugs that are also being evaluated in cancer. Studies with pitavastatin in primary patient derived tumor cells and established PDA lines, revealed dose dependent growth inhibition. At the molecular level, pitavastatin induced expression of the cyclin dependent kinase (CDK) inhibitor p21 in a cholesterol independent manner, blocked repressive phosphorylation of the Retinoblastoma tumor suppressor protein at CDK targeted sites, and reduced expression of E2F target genes required for progression through the G1/S boundary. Together, the data provide new insight into mechanisms by which statins constrain proliferation in cancer and establish the effectiveness of a novel screening platform to identify small molecules of clinical relevance in pancreatic cancer.