SUMMARY Early full-term pregnancy is one of the most effective natural protections against breast cancer. To investigate this effect, we have characterized the global gene expression and epigenetic profiles of multiple cell types from normal breast tissue of nulliparous and parous women, and carriers of BRCA1 or BRCA2 mutations. We found significant differences in CD44+ progenitor cells, where the levels of many stem cell-related genes and pathways, including the cell cycle regulator p27, are lower in parous women without BRCA1/BRCA2 mutations. We also noted a significant reduction in the frequency of CD44+p27+ cells in parous women, and showed using explant cultures that parity-related signaling pathways play a role in regulating the number of p27+ cells and their proliferation. Our results suggest that pathways controlling p27+ mammary epithelial cells and the numbers of these cells relate to breast cancer risk, and can be explored for cancer risk assessment and prevention.

The transcription factor Meis1 is expressed preferentially in hematopoietic stem cells (HSCs) and overexpressed in certain leukemias. However, the functions of Meis1 in hematopoiesis remain largely unknown. In the present study, we found that Meis1 is required for the maintenance of hematopoiesis under stress and over the long term, whereas steadystate hematopoiesis was sustained in the absence of Meis1 in inducible knock-out mice. BM cells of Meis1-deficient mice showed reduced colony formation and contained significantly fewer numbers of long-term HSCs, which exhibited loss of quiescence. Further, we found that Meis1 deletion led to the accumulation of reactive oxygen species in HSCs and decreased expression of genes implicated in hypoxia response. Finally, reac- IntroductionThe transcription factor Meis1 belongs to the 3-amino acid loop extension (TALE) family of homeodomain proteins, which also includes Pbx1. Meis1 was originally discovered as a target of activation by retroviral integration in the leukemic mouse strain BXH-2. In these mice, Meis1 was always found coactivated with Hoxa7 or Hoxa9. 1 Experimental overexpression of Meis1 and Hoxa9 in hematopoietic cells leads to an aggressive leukemia in mice. 2 In normal hematopoiesis, Meis1 expression among the various cell compartments is correlated with the degree of selfrenewal, with the levels being highest in hematopoietic stem cells (HSCs) and declining with differentiation. 3,4 These results, combined with those from studies in leukemia, suggest a role for Meis1 in HSC self-renewal. However, the functions of Meis1 in established hematopoiesis remain unknown. Meis1 Ϫ/Ϫ mice die by embryonic day 14.5 and the embryos display extensive hemorrhaging, particularly in the CNS. In addition, fetal liver cells obtained from Meis1 Ϫ/Ϫ embryos show reduced myeloid colony formation in vitro and perform poorly in competitive transplantation assays in vivo. 5,6 These results show that Meis1 is critical in the development of hematopoiesis. Further, Meis1 Ϫ/Ϫ embryos display defects in capillary formation, suggesting additional roles for Meis1 in mechanisms that regulate the process of angiogenesis, at least during development.Previous studies showed that Meis1 and Pbx1 can form heterodimeric and heterotrimeric complexes with HOX proteins, augmenting the binding and activation of target genes by HOX proteins such as HOXA9. 7,8 Deletion of Pbx1 in the hematopoietic system led to loss of quiescence in HSCs, resulting in hematopoietic failure. 9 Given that both Pbx1 and Meis1 are expressed in HSCs, we hypothesized that Meis1 might also be required for the maintenance of the self-renewing HSCs. In the present study, we investigated the role of Meis1 in established hematopoiesis using inducible knock-out mice. We found that Meis1 is required for the maintenance of HSCs by preserving quiescence in these mice. Maintenance of quiescence is critical for preserving self-renewal of long-term stem cells, and various signaling pathways have been shown to modulate quiescence in HSC...

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Purpose Classical pharmacology allows the use and development of conventional phytomedicine faster and more economically than conventional drugs. This approach should be tested for their efficacy in terms of complementarity and disease control. The purpose of this study was to determine the molecular mechanisms by which nimbolide, a triterpenoid found in the well-known medicinal plant Azadirachta indica controls glioblastoma (GBM) growth. Experimental Design Using in vitro signaling, anchorage-independent growth, kinase assays, and xenograft models, we investigated the mechanisms of its growth inhibition in glioblastoma. Results We show that nimbolide or an ethanol soluble fraction of A. indica leaves (Azt) that contains nimbolide as the principal cytotoxic agent is highly cytotoxic against GBM in vitro and in vivo. Azt caused cell cycle arrest, most prominently at the G1-S stage in GBM cells expressing EGFRvIII, an oncogene present in about 20-25% of GBMs. Azt/nimbolide directly inhibited CDK4/CDK6 kinase activity leading to hypophosphorylation of the retinoblastoma (RB) protein, cell cycle arrest at G1-S and cell death. Independent of RB hypophosphorylation, Azt also significantly reduced proliferative and survival advantage of GBM cells in vitro and in tumor xenografts by downregulating Bcl2 and blocking growth factor induced phosphorylation of Akt, Erk1/2 and STAT3. These effects were specific since Azt did not affect mTOR or other cell cycle regulators. In vivo, Azt completely prevented initiation and inhibited progression of GBM growth. Conclusions Our preclinical findings demonstrate Nimbolide as a potent anti-glioma agent that blocks cell cycle and inhibits glioma growth in vitro and in vivo.

Despite growing awareness of the biologic features underlying MLL-rearranged leukemia, targeted therapies for this leukemia have remained elusive and clinical outcomes remain dismal. MBNL1, a protein involved in alternative splicing, is consistently overexpressed in MLL-rearranged leukemias. We found that MBNL1 loss significantly impairs propagation of murine and human MLL-rearranged leukemia in vitro and in vivo. Through transcriptomic profiling of our experimental systems, we show that in leukemic cells, MBNL1 regulates alternative splicing (predominantly intron exclusion) of several genes including those essential for MLL-rearranged leukemogenesis, such as DOT1L and SETD1A. We finally show that selective leukemic cell death is achievable with a small molecule inhibitor of MBNL1. These findings provide the basis for a new therapeutic target in MLL-rearranged leukemia and act as further validation of a burgeoning paradigm in targeted therapy, namely the disruption of cancer-specific splicing programs through the targeting of selectively essential RNA binding proteins.

Recently, macroautophagy/autophagy has emerged as a promising target in various types of solid tumor treatment. However, the impact of autophagy on acute myeloid leukemia (AML) maintenance and the validity of autophagy as a viable target in AML therapy remain unclear. Here we show that Kmt2a/Mll-Mllt3/Af9 AML (MA9-AML) cells have high autophagy flux compared with normal bone marrow cells, but autophagy-specific targeting, either through Rb1cc1-disruption to abolish autophagy initiation, or via Atg5-disruption to prevent phagophore (the autophagosome precursor) membrane elongation, does not affect the growth or survival of MA9-AML cells, either in vitro or in vivo. Mechanistically, neither Atg5 nor Rb1cc1 disruption impairs endolysosome formation or survival signaling pathways. The autophagy inhibitor chloroquine shows autophagy-independent anti-leukemic effects in vitro but has no efficacy in vivo likely due to limited achievable drug efficacy in blood. Further, vesicular exocytosis appears to mediate chloroquine resistance in AML cells, and exocytotic inhibition significantly enhances the anti-leukemic effect of chloroquine. Thus, chloroquine can induce leukemia cell death in vitro in an autophagy-independent manner but with inadequate efficacy in vivo, and vesicular exocytosis is a possible mechanism of chloroquine resistance in MA9-AML. This study also reveals that autophagy-specific targeting is unlikely to benefit MA9-AML therapy.

• Meis1 is required for the maintenance of MLL-fusion gene leukemia; HLF is a key downstream mediator of Meis1.• Meis1 and HLF restrict oxidative stress; induction of oxidative phosphorylation may be therapeutic in leukemia.Leukemias with MLL translocations are often found in infants and are associated with poor outcomes. The pathogenesis of MLL-fusion leukemias has been linked to upregulation of HOX/MEIS1 genes. The functions of the Hox/Meis1 complex in leukemia, however, remain elusive. Here, we used inducible Meis1-knockout mice coupled with MLL-AF9 knockin mice to decipher the mechanistic role of Meis1 in established MLL leukemia. We demonstrate that Meis1 is essential for maintenance of established leukemia. In addition, in both the murine model and human leukemia cells, we found that Meis1 loss led to increased oxidative stress, oxygen flux, and apoptosis. Gene expression and chromatin immunoprecipitation studies revealed hepatic leukemia factor (HLF) as a target gene of Meis1. Hypoxia or HLF expression reversed the oxidative stress, rescuing leukemia development in Meis1-deficient cells. Thus, the leukemia-promoting properties of Meis1 are at least partly mediated by a lowoxidative state, aided by HLF. These results suggest that stimulants of oxidative metabolism could have therapeutic potential in leukemia treatment. (Blood. 2015;125(16):2544-2552 IntroductionReciprocal translocations of the 11q23 locus lead to acute leukemias of both myeloid and lymphoid lineages. These leukemias are usually resistant to conventional chemotherapies. The translocation generates an oncogenic fusion protein comprised of an amino terminus derived from MLL (now called KMT2A), the gene at 11q23, fused to a carboxy terminus derived from one of several different genes. Recent studies have revealed that these MLL-fusion proteins then join higher-order protein complexes containing various transcriptional activators and/or elongating factors such as DOT1L, PAFc, and p-TEFB. 1,2 Recruitment of these complexes to genomic loci was shown to be associated with chromatin modifications and subsequent changes in gene expression. 3 Regardless of the fusion partner involved in the translocation and of the consequential protein complex assembled, the downstream genes activated by the various MLL-fusion proteins are largely identical. Data from patientderived leukemia cells and from experimental models reveal that MLLfusion proteins upregulate the expression of posterior HOX-A genes and of the HOX cofactor MEIS1. [4][5][6] Further, retroviral overexpression of a Hox gene (with few exceptions) along with Meis1 in murine hematopoietic cells induces an aggressive leukemia in transplanted mice. 7,8 Thus, MLL-fusion protein induced upregulation of HOX and MEIS1 genes is considered central to the pathology of these leukemias.Studies to test the requirement of HOX-A and MEIS1 genes in MLL-fusion leukemias have yielded mixed results. Although shRNA knockdown of HOXA9 was shown to inhibit leukemia, mice lacking Hoxa9 protein developed leukemia induced...

Langerhans cell histiocytosis (LCH) is a rare hematologic neoplasm characterized by a clonal proliferation of Langerhans-like cells. Genomic profiling has identified recurrent somatic activating mutations in the mitogen-activated protein kinase pathway, which are targetable by small-molecule inhibitors. However, key questions such as the curative potential of targeted therapy and the cell of origin remain unanswered. In this study, we describe clinical outcomes of a series of pediatric patients with multisystem BRAF V600E–mutant LCH, as well as the results of accompanying murine xenograft experiments. Four infants with LCH (range, 7-11 months at diagnosis) and secondary hemophagocytic lymphohistiocytosis were referred to our institution and subsequently treated with the BRAF V600E–specific inhibitor dabrafenib. All patients achieved complete clinical responses by 8 weeks of therapy, with remissions lasting a median of 36 months (range, 27-42 months). One infant successfully discontinued therapy long-term upon achieving a molecular response by real-time quantitative polymerase chain reaction (RT-qPCR). We further characterized the disease-propagating cell population in a subset of these patients by transplanting whole bone marrow into immunodeficient mice. Xenografted animals exhibited decreased survival with hematologic abnormalities, splenomegaly, and histiocytic infiltrates in the bone marrow resembling human disease. This process could also be secondarily transplanted, resulting in a comparable disease latency with similar histologic findings. These data further support the presence of a disease-initiating cell in the bone marrow compartment. We demonstrate that despite aggressive disease behavior in a xenograft model, these patients can achieve sustained clinical remissions with targeted monotherapy, with a select subset achieving molecular responses by RT-qPCR.