Stephen Arnovitz

Although genomes encode instructions for mammalian cell differentiation with rich syntactic relationships, existing methods for genetically programming cells have only modest capabilities for stepwise gene regulation. Here, we develop a sequential genetic system that transcriptionally activates endogenous genes in a preprogrammed, stepwise manner. This system uses the removal of an RNA polymerase III termination sequence to trigger both the transcriptional activation and DNA endonuclease activities of a Cas9-VPR protein, driving progression through a cascade of gene activation events. The system’s functionality in human cells, including iPSCs, enables the development of a path for cellular programming by controlling the sequential order of gene activation to influence cellular states.

The diversity of regulatory T (T_reg) cells in health and in disease remains unclear. Individuals with colorectal cancer harbor a subpopulation of RORγt⁺ T_reg cells with elevated expression of β-catenin and pro-inflammatory properties. Here we show progressive expansion of RORγt⁺ T_reg cells in individuals with inflammatory bowel disease during inflammation and early dysplasia. Activating Wnt–β-catenin signaling in human and murine T_reg cells was sufficient to recapitulate the disease-associated increase in the frequency of RORγt⁺ T_reg cells coexpressing multiple pro-inflammatory cytokines. Binding of the β-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt–β-catenin activation induced newly accessible chromatin sites in these genes and upregulated their expression. These findings indicate that TCF-1 and Foxp3 together limit the expression of pro-inflammatory genes in T_reg cells. Activation of β-catenin signaling interferes with this function and promotes the disease-associated RORγt⁺ T_reg phenotype.

MicroRNAs (miRNAs) have been implicated in the pathogenesis of various types of cancers. We reported previously that miR-126 likely functioned as an oncogene in acute myeloid leukemia (AML) (Li Z, et al. PNAS. 2008 Oct 7; 105:15535-40), which was confirmed by others (de Leeuw DC, et al. Cancer Research. 2014 Apr 1,74:2094-105; Dorrance AM, et al. Leukemia. 2015). However, it was also reported that miR-126 knockdown in normal hematopoietic stem/progenitor cells (HSPCs) resulted in expansion of long-term HSCs (Lechman ER, et al. Cell Stem Cell. 2012 Dec 7,11:799-811). Thus, the role of miR-126 in normal and malignant hematopoiesis warrants further investigation. Here we show that miR-126 is particularly overexpressed in t(8;21) AML and increased expression of miR-126 is associated with poor prognosis in patients with t(8;21) AML. To determine the function of miR-126 in leukemogenesis, we conducted both gain- and loss-of-function in vivo studies of miR-126 in t(8;21) AML models via mouse bone marrow transplantation (BMT). Surprisingly, we found that both overexpression and knockout of miR-126 accelerated leukemogenesis by the t(8;21) fusion genes, AML1-ETO (AE) or AML1-ETO9a (AE9a; a potent oncogenic isoform of AE). For example, forced expression of miR-126 (by retroviral transduction?) significantly accelerated AE9a-mediated leukemogenesis (median survival of AE9a+miR-126 vs. AE9a: 234 days vs. 317 days, p AE9a -transduced miR-126^-/- HSPCs (miR-126KO+AE9a) caused leukemia significantly faster than did AE9a -transduced wild-type HSPCs (AE9a) in recipients (median survival: 130 days vs. 317 days, p vs. 81 days, p =0.22), and both groups had significantly shorter survival (p AE9a +miR-126 group developed leukemia significantly faster than did the miR-126KO+AE9a group in both tertiary and quaternary BMT (median survival: 28 days vs. 43 days, p =0.0001 in tertiary; 35 days vs. 49 days, p p p 10 fold), and both were significantly higher (p in vivo and in vitro studies demonstrated that depletion or inhibition of miR-126 significantly sensitized t(8;21)-fusion-induced mouse leukemia or primary human leukemia cells to standard chemotherapy. Our mechanistic studies indicate that miR-126 overexpression activates genes that are highly expressed in LSCs/LICs and/or primitive HSPCs through directly targeting ERRFI1 and SPRED 1, which in turn activate the MAPK signaling pathway. On the other hand, miR-126 knockout activates genes that are highly expressed in committed, more differentiated hematopoietic cells and triggers the WNT/β-catenin signaling pathway by inducing FZD7 expression. We show that ERRFI1, SPRED 1 and FZD7 are bona fide targets of miR-126, and their increased expression is associated favorable survival of t(8;21) AML patients. Overall, our data show that both gain- and loss-of-function of a single miRNA (e.g., miR-126) can promote tumorigenesis and enhance long-term self-renewal/progression of LSCs/LICs, through targeting distinct gene signaling and thus being associated with different biological consequences. As miR-126 depletion can sensitize AML cells to standard chemotherapy, our data also suggest that miR-126 represents a promising therapeutic target.