The miR-424(322)/503 cluster orchestrates remodeling of the epithelium in the involuting mammary gland.

The mammary gland is a very dynamic organ that undergoes continuous remodeling. The critical regulators of this process are not fully understood. Here we identify the microRNA cluster miR-424(322)/503 as an important regulator of epithelial involution after pregnancy. Through the generation of a knockout mouse model, we found that regression of the secretory acini of the mammary gland was compromised in the absence of miR-424(322)/503. Mechanistically, we show that miR-424(322)/503 orchestrates cell life and death decisions by targeting BCL-2 and IGF1R (insulin growth factor-1 receptor). Furthermore, we demonstrate that the expression of this microRNA cluster is regulated by TGF-ß, a well-characterized regulator of mammary involution. Overall, our data suggest a model in which activation of the TGF-ß pathway after weaning induces the transcription of miR-424(322)/503, which in turn down-regulates the expression of key genes. Here, we unveil a previously unknown, multilayered regulation of epithelial tissue remodeling coordinated by the microRNA cluster miR-424(322)/503.

A thrombopoietin receptor antagonist is capable of depleting myelofibrosis hematopoietic stem and progenitor cells.

Recently, interactions between thrombopoietin (TPO) and its receptor, the myeloproliferative leukemia (MPL) virus oncogene, have been shown to play a role in the development and progression of myeloproliferative neoplasms including myelofibrosis (MF). These observations have led to the development of strategies to disrupt the association of TPO with its receptor as a means of targeting MF hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). In this report, we show that although both splenic and peripheral blood MF CD34(+) cells expressed lower levels of MPL than normal CD34(+) cells, TPO promoted the proliferation of MF CD34(+) cells and HPCs in a dose-dependent fashion. Furthermore, the treatment of MF but not normal CD34(+) cells with a synthesized MPL antagonist, LCP4, decreased the number of CD34(+)Lin(-) cells and all classes of assayable HPCs (colony-forming unit-megakaryocyte [CFU-MK], CFU-granulocyte/macrophage, burst-forming unit-erythroid/CFU-erythroid, and CFU-granulocyte/erythroid/macrophage/MK) irrespective of their mutational status. In addition, LCP4 treatment resulted in the depletion of the number of MF HPCs that were JAK2V617F(+) Moreover, the degree of human cell chimerism and the proportion of malignant donor cells were significantly reduced in immunodeficient mice transplanted with MF CD34(+) cell grafts treated with LCP4. These effects of LCP4 on MF HSCs/HPCs were associated with inhibition of JAK-STAT activity, leading to the induction of apoptosis. These findings demonstrate that such specific anti-cytokine receptor antagonists represent a new class of drugs that are capable of targeting MF HSCs.

Antitumor T-cell Homeostatic Activation Is Uncoupled from Homeostatic Inhibition by Checkpoint Blockade.

T-cell transfer into lymphodepleted recipients induces homeostatic activation and potentiates antitumor efficacy. In contrast to canonical T-cell receptor-induced activation, homeostatic activation yields a distinct phenotype and memory state whose regulatory mechanisms are poorly understood. Here, we show in patients and murine models that, following transfer into lymphodepleted bone marrow transplant (BMT) recipients, CD8+ T cells undergo activation but also simultaneous homeostatic inhibition manifested by upregulation of immune-checkpoint molecules and functional suppression. T cells transferred into BMT recipients were protected from homeostatic inhibition by PD-1/CTLA4 dual checkpoint blockade (dCB). This combination of dCB and BMT-"immunotransplant"-increased T-cell homeostatic activation and antitumor T-cell responses by an order of magnitude. Like homeostatic activation, homeostatic inhibition is IL7/IL15-dependent, revealing mechanistic coupling of these two processes. Marked similarity in ex vivo modulation of post-BMT T cells in mice and patients is promising for the clinical translation of immunotransplant (NCT03305445) and for addressing homeostatic inhibition in T-cell therapies. SIGNIFICANCE: For optimal anticancer effect, T-cell therapies including chimeric antigen receptor T-cell, tumor-infiltrating lymphocyte, and transgenic T-cell therapies require transfer into lymphodepleted recipients and homeostatic activation; however, concomitant homeostatic inhibition mitigates T-cell therapies' efficacy. Checkpoint blockade uncouples homeostatic inhibition from activation, amplifying T-cell responses. Conversely, tumors nonresponsive to checkpoint blockade or BMT are treatable with immunotransplant.See related commentary by Ansell, p. 1487.This article is highlighted in the In This Issue feature, p. 1469.

The microRNA 424/503 cluster reduces CDC25A expression during cell cycle arrest imposed by transforming growth factor ß in mammary epithelial cells.

Recently, we demonstrated that the microRNA 424(322)/503 [miR-424(322)/503] cluster is transcriptionally controlled by transforming growth factor ß (TGF-ß) in the mammary epithelium. Induction of this microRNA cluster impacts mammary epithelium fate by regulating apoptosis and insulin-like growth factor 1 (IGF1) signaling. Here, we expanded our finding to demonstrate that miR-424(322)/503 is an integral component of the cell cycle arrest mediated by TGF-ß. Mechanistically, we showed that after TGF-ß exposure, increased levels of miR-424(322)/503 reduce the expression of the cell cycle regulator CDC25A. miR-424(322)/503-dependent posttranscriptional downregulation of CDC25A cooperates with previously described transcriptional repression of the CDC25A promoter and proteasome-mediated degradation to reduce the levels of CDC25A expression and to induce cell cycle arrest. We also provide evidence that the TGF-ß/miR-424(322)/503 axis is part of the mechanism that regulates the proliferation of hormone receptor-positive (HR(+)) mammary epithelial cells in vivo.

Pooled shRNA screenings: experimental approach.

RNA interference (RNAi) has emerged as a powerful genetic strategy to functionally interrogate the entire genome by loss-of-function studies. In the last years, several arrayed shRNA libraries that can silence almost all the human genome have been developed. The generation of new and more efficient shRNA plasmids has allowed performing genetic screens in a pooled fashion and provides the opportunity to investigate the entire genome finding relevant genes to any biological process. In this chapter, the pipeline and methods to perform a pooled shRNA screen are discussed.