Freshwater planarians are well known for their regenerative abilities. Less well known is how planarians maintain spatial patterning in long-lived adult animals or how they re-pattern tissues during regeneration. HOX genes are good candidates to regulate planarian spatial patterning, yet the full complement or genomic clustering of planarian HOX genes has not yet been described, primarily because only a few have been detectable by in situ hybridization, and none have given morphological phenotypes when knocked down by RNAi. Because the planarian Schmidtea mediterranea (S. mediterranea) is unsegmented, appendage less, and morphologically simple, it has been proposed that it may have a simplified HOX gene complement. Here, we argue against this hypothesis and show that S. mediterranea has a total of 13 HOX genes, which represent homologs to all major axial categories, and can be detected by whole-mount in situ hybridization using a highly sensitive method. In addition, we show that planarian HOX genes do not cluster in the genome, yet 5/13 have retained aspects of axially restricted expression. Finally, we confirm HOX gene axial expression by RNA deep-sequencing 6 anterior–posterior “zones” of the animal, which we provide as a dataset to the community to discover other axially restricted transcripts. Freshwater planarians have an unappreciated HOX gene complexity, with all major axial categories represented. However, we conclude based on adult expression patterns that planarians have a derived body plan and their asexual lifestyle may have allowed for large changes in HOX expression from the last common ancestor between arthropods, flatworms, and vertebrates. Using our in situ method and axial zone RNAseq data, it should be possible to further understand the pathways that pattern the anterior–posterior axis of adult planarians.

Triple-negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which no specific treatment is currently available. Although the retinoblastoma tumor-suppressor gene (RB1) is frequently lost together with TP53 in TNBC, it is not directly targetable. There is thus great interest in identifying vulnerabilities downstream of RB1 that can be therapeutically exploited. Here, we determined that combined inactivation of murine Rb and p53 in diverse mammary epithelial cells induced claudin-low–like TNBC with Met, Birc2/3-Mmp13-Yap1, and Pvt1-Myc amplifications. Gene set enrichment analysis revealed that Rb/p53-deficient tumors showed elevated expression of the mitochondrial protein translation (MPT) gene pathway relative to tumors harboring p53 deletion alone. Accordingly, bioinformatic, functional, and biochemical analyses showed that RB1-E2F complexes bind to MPT gene promoters to regulate transcription and control MPT. Additionally, a screen of US Food and Drug Administration–approved (FDA-approved) drugs identified the MPT antagonist tigecycline (TIG) as a potent inhibitor of Rb/p53-deficient tumor cell proliferation. TIG preferentially suppressed RB1-deficient TNBC cell proliferation, targeted both the bulk and cancer stem cell fraction, and strongly attenuated xenograft growth. It also cooperated with sulfasalazine, an FDA-approved inhibitor of cystine xCT antiporter, in culture and xenograft assays. Our results suggest that RB1 deficiency promotes cancer cell proliferation in part by enhancing mitochondrial function and identify TIG as a clinically approved drug for RB1-deficient TNBC.

The tumor suppressor PTEN is frequently inactivated in breast and other cancers; yet, germ-line mutations in this gene induce nonmalignant hamartomas, indicating dependency on additional cooperating events. Here we show that most tumors derived from conditional deletion of mouse pten in mammary epithelium are highly differentiated and lack transplantable tumor-initiating cells (TICs) capable of seeding new tumors following orthotopic injection of FACS-sorted or tumorsphere cells. A rare group of poorly differentiated tumors did harbor transplantable TICs. These transplantable tumors exhibited distinct molecular classification, signaling pathways, chromosomal aberrations, and mutational landscape, as well as reduced expression of microRNA-143/145 (miR-143/145). Stable knockdown of miR-143/145 conferred tumorigenic potential upon poorly transplantable pten -deficient tumor cells through a mechanism involving induction of RAS signaling, leading to increased sensitivity to MEK inhibition. In humans, miR-145 deficiency significantly correlated with elevated RAS-pathway activity in basal-like breast cancer, and patients with combined PTEN/miR-145 loss or PTEN-loss/high RAS-pathway activity exhibited poor clinical outcome. These results underscore a selective pressure for combined PTEN loss together with RAS-pathway activation, either through miR-145 loss or other mechanisms, in basal-like breast cancer, and a need to identify and prioritize these tumors for aggressive therapy.

Hematopoietic homeostasis is tightly regulated by controlling the balance between quiescence, self-renewal, and lineage-commitment of hematopoietic stem cells (HSCs). Deregulation of these processes and aberrant acquisition of stem cell-like properties is believed to be central to the pathogenesis of hematologic malignancies, such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, little is known about the molecular networks maintaining the stem cell state and the epigenetic and post-transcriptional regulation of determinants that control these programs. MicroRNAs (miRNAs) represent a large class of post-transcriptional regulators that mediate repression of multiple target mRNAs. We have previously shown that miR-126 and miR-125a are differentially expressed across the human hematopoietic hierarchy and function to control self-renewal and cell fate decisions by reinforcing gene expression programs in a developmental stage-specific manner (Lechman et al. Cell Stem Cell, 2012; Wojtowitz et al. Cell Stem Cell, 2016). To identify additional miRNA(s) that play a functional role in hematopoiesis, we performed an in vivo competitive repopulation screen in which candidate miRNAs were overexpressed (OE) in human CD34+CD38- umbilical cord blood (CB) cells and subsequently transplanted into immune-deficient mice for 24 weeks. miR-130a was shown to enhance long-term hematopoietic reconstitution and chosen for further investigation. At 12 and 24 weeks after transplantation, enforced miR-130a expression (including an mOrange-mO+ indicator) conferred a competitive advantage over untransduced CB cells demonstrated by increased CD45+ human chimerism in the injected femur (IF), bone marrow (BM), and spleen of recipient mice. miR-130 enforced expression (miR-130a OE) increased the proportion of mO+/hCD45+ cells by approximately 2- and 5-fold after 12 and 24 weeks of repopulation, respectively. miR-130a OE xenografts showed multilineage engraftment with increased myeloid lineage output and significantly enhanced erythroid development at the expense of B-lymphoid lineage output in BM and spleen of recipient mice. Detailed flow cytometry analysis of xenografts revealed accumulation of immature GlyA+/CD71+/CD36+ erythroid progenitors, suggesting a differentiation block at the polychromic erythroblast stage. Notably, miR-130a OE induced the expansion of CD34+CD38- Lin- compartment and increased proportion of CD34+CD38-CD90+CD45RA- immuno-phenotypic HSC. Secondary transplantation involving limiting dilution analysis revealed approximately a 10-fold increase in HSC frequency, consistent with a role of miR-130a in HSC self-renewal. The lineage potential of miR-130OE primitive cells was assessed in vitro using single-cell stromal-based myelo-erythroid differentiation assay. Enforced expression of miR-130a in human HSC and multipotent progenitors (MPP) resulted in the decreased frequency of unipotent myeloid output (M colonies) and increased multipotent output (M/E/Meg, E/Meg colonies), supporting a role of miR-130a in erythroid-megakaryocytic fate specification. Label-free semiquantitative proteomics and subsequent gene set enrichment pathway analysis (GSEA) were performed on miR-130a OE and control transduced CD34+ CB cells to elucidate molecular mechanism(s) of miR-130a function. We identified that miR-130a modulated pathways centered on translational regulation and chromatin modification. Together, our data suggest that miR-130a plays a role in the regulation of the HSC self-renewal and erythroid differentiation. Given that several studies showed aberrant expression of miR-130a in MDS and some AML subtypes, it is important to delineate the role of miR-130a in normal hematopoiesis to comprehend its potential contribution to the development of hematologic malignancies. This abstract is also being presented as Poster 40. Citation Format: Gabriela Krivdova, Eric R. Lechman, Erwin M. Schoof, Veronique Voisin, Olga I. Gan, Aaron Trotman-Grant, Karin G. Hermans, Gary D. Bader, John E. Dick. MicroRNA-130a regulates hematopoietic stem cell self-renewal and erythroid differentiation [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR07.