Insulin receptor loss impairs mammary tumorigenesis in mice.

Breast cancer (BC) prognosis and outcome are adversely affected by obesity. Hyperinsulinemia, common in the obese state, is associated with higher risk of death and recurrence in BC. Up to 80% of BCs overexpress the insulin receptor (INSR), which correlates with worse prognosis. INSR's role in mammary tumorigenesis was tested by generating MMTV-driven polyoma middle T (PyMT) and ErbB2/Her2 BC mouse models, respectively, with coordinate mammary epithelium-restricted deletion of INSR. In both models, deletion of either one or both copies of INSR leads to a marked delay in tumor onset and burden. Longitudinal phenotypic characterization of mouse tumors and cells reveals that INSR deletion affects tumor initiation, not progression and metastasis. INSR upholds a bioenergetic phenotype in non-transformed mammary epithelial cells, independent of its kinase activity. Similarity of phenotypes elicited by deletion of one or both copies of INSR suggest a dose-dependent threshold for INSR impact on mammary tumorigenesis.

Ultrasensitive Detection of NOTCH1 c.7544_7545delCT Mutations in Chronic Lymphocytic Leukemia by Droplet Digital PCR Reveals High Frequency of Subclonal Mutations and Predicts Clinical Outcome in Cases with Trisomy 12.

NOTCH1 is recurrently mutated in chronic lymphocytic leukemia (CLL), most commonly as a 2-bp frameshift deletion (c.7541_7542delCT). This mutated allele encodes a truncated form of the receptor (p.P2514Rfs∗4) lacking the C-terminal proline, glutamic acid, serine, and threonine (PEST) degradation domain that increases NOTCH1 signaling duration. NOTCH1 mutation has been associated with poor clinical outcomes in CLL. We validated a highly sensitive and quantitative droplet digital PCR assay for the NOTCH1 delCT mutation, which was anticipated to perform well compared with Sanger sequencing and allele-specific PCR. Performance characteristics of this assay were tested on 126 samples from an unselected CLL cohort and a separate cohort of 85 samples from patients with trisomy 12 CLL. The delCT mutation was detected at allele frequencies as low as 0.024%; 25% of unselected cases and 55% of trisomy 12 cases were positive at the 0.024% detection threshold. Mutational burdens ≥1% were significantly associated with shorter overall survival (OS) in patients with trisomy 12+ disease in multivariate analysis (median OS, 9.1 versus 13 years, with hazard ratio of 2.34; P = 0.031). Mutational burdens <1% correlated with shorter OS in univariate, but not multivariate, analyses. These results suggest that droplet digital PCR testing for NOTCH1 delCT mutation may aid in risk stratification and/or disease monitoring in certain subsets of patients with CLL.

Population-based phase II trial of stereotactic ablative radiotherapy (SABR) for up to 5 oligometastases: SABR-5.

BACKGROUND: Oligometastases refer to a state of disease where cancer has spread beyond the primary site, but is not yet widely metastatic, often defined as 1-3 or 1-5 metastases in number. Stereotactic ablative radiotherapy (SABR) is an emerging radiotherapy technique to treat oligometastases that require further prospective population-based toxicity estimates. METHODS: This is a non-randomized phase II trial where all participants will receive experimental SABR treatment to all sites of newly diagnosed or progressing oligometastatic disease. We will accrue 200 patients to assess toxicity associated with this experimental treatment. The study was powered to give a 95% confidence on the risk of late grade 4 toxicity, anticipating a < 5% rate of grade 4 toxicity. DISCUSSION: SABR treatment of oligometastases is occurring off-trial at a high rate, without sufficient evidence of its efficacy or toxicity. This trial will provide necessary toxicity data in a population-based cohort, using standardized doses and organ at risk constraints, while we await data on efficacy from randomized phase III trials. TRIAL REGISTRATION: Registered through clinicaltrials.gov NCT02933242 on October 14, 2016 prospectively before patient accrual.

Epigenetic Restoration of Fetal-like IGF1 Signaling Inhibits Leukemia Stem Cell Activity.

Acute leukemias are aggressive malignancies of developmentally arrested hematopoietic progenitors. We sought here to explore the possibility that changes in hematopoietic stem/progenitor cells during development might alter the biology of leukemias arising from this tissue compartment. Using a mouse model of acute T cell leukemia, we found that leukemias generated from fetal liver (FL) and adult bone marrow (BM) differed dramatically in their leukemia stem cell activity with FL leukemias showing markedly reduced serial transplantability as compared to BM leukemias. We present evidence that this difference is due to NOTCH1-driven autocrine IGF1 signaling, which is active in FL cells but restrained in BM cells by EZH2-dependent H3K27 trimethylation. Further, we confirmed this mechanism is operative in human disease and show that enforced IGF1 signaling effectively limits leukemia stem cell activity. These findings demonstrate that resurrecting dormant fetal programs in adult cells may represent an alternate therapeutic approach in human cancer.

IGF1R Derived PI3K/AKT Signaling Maintains Growth in a Subset of Human T-Cell Acute Lymphoblastic Leukemias.

Insulin-like growth factor 1 receptor (IGF1R) is a prevalent signaling pathway in human cancer that supports cell growth/survival and thus contributes to aggressive biological behavior. Much work has gone into development of IGF1R inhibitors; however, candidate agents including small molecule tyrosine kinase inhibitors and blocking antibodies have yet to fulfill their promise clinically. Understanding cellular features that define sensitivity versus resistance are important for effective patient selection and anticipation of outgrowth of a resistant clone. We previously identified an important role for IGF signaling in T-cell acute lymphoblastic leukemia (T-ALL) relying primarily upon genetically defined mouse models. We present here an assessment of IGF1R dependence in human T-ALL using a broad panel of 27 established cell lines that capture a spectrum of the genetic variation that might be encountered in clinical practice. We observed that a subset of cell lines are sensitive to IGF1R inhibition and are characterized by high levels of surface IGF1R expression and PTEN positivity. Interestingly, lentiviral expression or knock-down of PTEN in PTEN-negative/positive cell lines, respectively, had limited effects on their response to IGF1R inhibition, suggesting that PTEN contributes to, but does not define IGF dependence. Additionally, we characterize downstream PI3K/AKT signaling as dominant over RAS/RAF/MEK/ERK in mediating growth and/or survival in this context. Finally, we demonstrate that IGF and interleukin-7 (IL-7) fulfill non-overlapping roles in supporting T-ALL growth. These findings are significant in that they reveal cellular features and downstream mechanisms that may determine the response of an individual patient's tumor to IGF1R inhibitor therapy.

CD44 promotes chemoresistance in T-ALL by increased drug efflux.

T-Cell acute lymphoblastic leukemia is considered a largely curable disease in children; however, adult patients and children with refractory or relapsed disease have consistently poor outcomes. On the basis of our prior work highlighting CD44 as a marker of leukemia-initiating cells in animal models and because cancer stem cells are postulated to possess intrinsic resistance to conventional chemotherapy, we examined whether CD44 itself might play a role in mediating chemoresistance. We report here that in both genetically defined mouse models and human cell lines, CD44 expression is associated with chemoresistance, and that this effect is mediated in part through enhanced drug efflux. Interestingly, we also observed increased CD44 expression in residual blasts following standard induction chemotherapy, as compared with blasts from matched, pretherapy samples in a subset of pediatric patients undergoing minimal residual disease monitoring as part of a clinical trial. These findings support a functional role for CD44 in promoting chemotherapy resistance and suggest that targeting it directly or its relevant effector pathways may improve clinical responses in T-cell acute lymphoblastic leukemia.

Leukemia stem cells in T-ALL require active Hif1α and Wnt signaling.

The Wnt signaling pathway has been shown to play important roles in normal hematopoietic stem cell biology and in the development of both acute and chronic myelogenous leukemia. Its role in maintaining established leukemia stem cells, which are more directly relevant to patients with disease, however, is less clear. To address what role Wnt signaling may play in T-cell acute lymphoblastic leukemia (T-ALL), we used a stably integrated fluorescent Wnt reporter construct to interrogate endogenous Wnt signaling activity in vivo. In this study, we report that active Wnt signaling is restricted to minor subpopulations within bulk tumors, that these Wnt-active subsets are highly enriched for leukemia-initiating cells (LICs), and that genetic inactivation of ß-catenin severely reduces LIC frequency. We show further that ß-catenin transcription is upregulated by hypoxia through hypoxia-inducible factor 1α (Hif1α) stabilization, and that deletion of Hif1α also severely reduces LIC frequency. Of note, the deletion of ß-catenin or Hif1α did not impair the growth or viability of bulk tumor cells, suggesting that elements of the Wnt and Hif pathways specifically support leukemia stem cells. We also confirm the relevance of these findings to human disease using cell lines and patient-derived xenografts, suggesting that targeting these pathways could benefit patients with T-ALL.

Inducible and coupled expression of the polyomavirus middle T antigen and Cre recombinase in transgenic mice: an in vivo model for synthetic viability in mammary tumour progression.

INTRODUCTION: Effective in vivo models of breast cancer are crucial for studying the development and progression of the disease in humans. We sought to engineer a novel mouse model of polyomavirus middle T antigen (PyV mT)-mediated mammary tumourigenesis in which inducible expression of this well-characterized viral oncoprotein is coupled to Cre recombinase (TetO-PyV mT-IRES-Cre recombinase or MIC). METHODS: MIC mice were crossed to the mouse mammary tumour virus (MMTV)-reverse tetracycline transactivator (rtTA) strain to generate cohorts of virgin females carrying one or both transgenes. Experimental (rtTA/MIC) and control (rtTA or MIC) animals were administered 2 mg/mL doxycycline beginning as early as eight weeks of age and monitored for mammary tumour formation, in parallel with un-induced controls of the same genotypes. RESULTS: Of the rtTA/MIC virgin females studied, 90% developed mammary tumour with complete penetrance to all glands in response to doxycycline and a T50 of seven days post-induction, while induced or un-induced controls remained tumour-free after one year of induction. Histological analyses of rtTA/MIC mammary glands and tumour revealed that lesions followed the canonical stepwise progression of PyV mT tumourigenesis, from hyperplasia to mammary intraepithelial neoplasia/adenoma, carcinoma, and invasive carcinoma that metastasizes to the lung; at each of these stages expression of PyV mT and Cre recombinase transgenes was confirmed. Withdrawal of doxycycline from rtTA/MIC mice with end-stage mammary tumours led to rapid regression, yet animals eventually developed PyV mT-expressing and -non-expressing recurrent masses with varied tumour histopathologies. CONCLUSIONS: We have successfully created a temporally regulated mouse model of PyV mT-mediated mammary tumourigenesis that can be used to study Cre recombinase-mediated genetic changes simultaneously. While maintaining all of the hallmark features of the well-established constitutive MMTV-PyV mT model, the utility of this strain derives from the linking of PyV mT and Cre recombinase transgenes; mammary epithelial cells are thereby forced to couple PyV mT expression with conditional ablation of a given gene. This transgenic mouse model will be an important research tool for identifying synthetic viable genetic events that enable PyV mT tumours to evolve in the absence of a key signaling pathway.

NOTCH1 promotes T cell leukemia-initiating activity by RUNX-mediated regulation of PKC-θ and reactive oxygen species.

Reactive oxygen species (ROS), a byproduct of cellular metabolism, damage intracellular macromolecules and, when present in excess, can promote normal hematopoietic stem cell differentiation and exhaustion. However, mechanisms that regulate the amount of ROS in leukemia-initiating cells (LICs) and the biological role of ROS in these cells are largely unknown. We show here that the ROS(low) subset of CD44(+) cells in T cell acute lymphoblastic leukemia (T-ALL), a malignancy of immature T cell progenitors, is highly enriched in the most aggressive LICs and that ROS accumulation is restrained by downregulation of protein kinase C θ (PKC-θ). Notably, primary mouse T-ALLs lacking PKC-θ show improved LIC activity, whereas enforced PKC-θ expression in both mouse and human primary T-ALLs compromised LIC activity. We also show that PKC-θ is regulated by a new pathway in which NOTCH1 induces runt-related transcription factor 3 (RUNX3), RUNX3 represses RUNX1 and RUNX1 induces PKC-θ. NOTCH1, which is frequently activated by mutation in T-ALL and required for LIC activity in both mouse and human models, thus acts to repress PKC-θ. These results reveal key functional roles for PKC-θ and ROS in T-ALL and suggest that aggressive biological behavior in vivo could be limited by therapeutic strategies that promote PKC-θ expression or activity, or the accumulation of ROS.

IGF signaling contributes to malignant transformation of hematopoietic progenitors by the MLL-AF9 oncoprotein.

Malignant transformation of normal hematopoietic progenitors is a multistep process that likely requires interaction between collaborating oncogenic signals at critical junctures. For instance, the MLL-AF9 fusion oncogene is thought to contribute to myeloid leukemogenesis by driving a hematopoietic stem cell-like "self-renewal" gene expression signature in committed myeloid progenitors. In addition, insulin-like growth factor (IGF) signaling has been implicated in self-renewal/pluripotency in hematopoietic and embryonic stem cell contexts and supports cell growth/survival by activation of downstream pathways, including phosphatidylinositol 3-kinase/Akt and Ras/Raf/extracellular signal-regulated kinase. We hypothesized that IGF signaling could be an important contributor in the process of cellular transformation and/or clonal propagation. Utilizing an MLL-AF9 mouse bone marrow transplantation model of acute myelogenous leukemia, we discovered that committed myeloid progenitor cells with genetically reduced levels of IGF1R were less susceptible to leukemogenic transformation due, at least in part, to a cell-autonomous defect in clonogenic activity. Rather unexpectedly, genetic deletion of IGF1R by inducible Cre recombinase had no effect on growth/survival of established leukemia cells. These findings suggest that IGF1R signaling contributes to transformation of normal myeloid progenitor cells, but is not required for propagation of the leukemic clone once it has become established. We also show that treatment of mouse MLL-AF9 acute myelogenous leukemia cells with BMS-536924, an IGF1R/insulin receptor-selective tyrosine kinase inhibitor, blocked cell growth, suggesting its efficacy in this model may be due to inhibition of insulin receptor and/or related tyrosine kinases, and raising the possibility that similar IGF1R inhibitors in clinical development may be acting through alternate/related pathways.

Receptor tyrosine kinase signaling favors a protumorigenic state in breast cancer cells by inhibiting the adaptive immune response.

Using transgenic mouse models of breast cancer that ablate Src homology and collagen A (ShcA) expression or oncogene-coupled ShcA signaling, we previously showed that this adaptor is critical for mammary tumor onset and progression. We now provide the first evidence that ShcA regulates mammary tumorigenesis, in part, through its ability to regulate the adaptive immune response. Inactivation of ShcA signaling within tumor cells results in extensive CD4(+) T-cell infiltration and induction of a humoral immune response in mammary tumors. This is associated with a robust CTL response in preneoplastic lesions that are deficient in ShcA signaling. Moreover, mammary tumor progression of ShcA-deficient hyperplasias is accelerated in a T cell-deficient background. We also uncover a clinically relevant correlation between high ShcA expression and low CTL infiltration in human breast cancers. Finally, we define a novel ShcA-regulated immune signature that functions as an independent prognostic marker of survival in human epidermal growth factor receptor 2(+) and basal breast cancers. We reveal a novel role for tumor cell-derived ShcA in the establishment and maintenance of an immunosuppressive state.

ShcA signalling is essential for tumour progression in mouse models of human breast cancer.

To explore the in vivo significance of ShcA during mammary tumorigenesis, we used mice expressing several phosphotyrosine-deficient ShcA alleles under the control of their endogenous promoter. We show that all three ShcA tyrosine phosphorylation sites are involved in the early stages of mammary tumour progression, including loss of the myoepithelial cell layer surrounding hyperplasias and during progression to carcinoma. We have determined that signals emanating from Y313 are important for tumour cell survival, whereas Y239/240 transduce signals promoting tumour vascularization. We further demonstrate that loss of ShcA expression in mammary epithelial cells abrogates tumour development. This study is the first to directly demonstrate that signalling downstream from the ShcA adaptor protein is critical for breast cancer development.

Distinct ErbB-2 coupled signaling pathways promote mammary tumors with unique pathologic and transcriptional profiles.

ErbB-2 overexpression and amplification occurs in 15% to 30% of human invasive breast carcinomas associated with poor clinical prognosis. Previously, we have shown that four ErbB-2/Neu tyrosine-autophosphorylation sites within the cytoplasmic tail of the receptor recruit distinct adaptor proteins and are sufficient to mediate transforming signals in vitro. Two of these sites, representing the growth factor receptor binding protein 2 (Grb2; Neu-YB) and the Src homology and collagen (Shc; Neu-YD) binding sites, can induce mammary tumorigenesis and metastasis. Here, we show that transgenic mice bearing the two other ErbB-2 autophosphorylation sites (Neu-YC and Neu-YE) develop metastatic mammary tumors. A detailed comparison of biological profiles among all Neu mutant mouse models revealed that Neu-YC, Neu-YD, and Neu-YE mammary tumors shared similar pathologic and transcriptional features. By contrast, the Neu-YB mouse model displayed a unique pathology with a high metastatic potential that correlates with a distinct transcriptional profile, including genes that promote malignant tumor progression such as metalloproteinases and chemokines. Furthermore, Neu-YB tumor epithelial cells showed abundant intracellular protein level of the chemokine CXCL12/SDF-1alpha, which may reflect the aggressive nature of this Neu mutant mouse model. Taken together, these findings indicate that activation of distinct Neu-coupled signaling pathways has an important impact on the biological behavior of Neu-induced tumors.