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Supplementary Figures 1-9 from Receptor Tyrosine Kinase Signaling Favors a Protumorigenic State in Breast Cancer Cells by Inhibiting the Adaptive Immune Response

Supplementary Tables 1-6 from Receptor Tyrosine Kinase Signaling Favors a Protumorigenic State in Breast Cancer Cells by Inhibiting the Adaptive Immune Response

Supplementary Figures 10-13 from Receptor Tyrosine Kinase Signaling Favors a Protumorigenic State in Breast Cancer Cells by Inhibiting the Adaptive Immune Response

Supplementary Methods and Figure Legends 1-13 from Receptor Tyrosine Kinase Signaling Favors a Protumorigenic State in Breast Cancer Cells by Inhibiting the Adaptive Immune Response

Breast cancer (BC) prognosis and outcome are adversely affected by increased body weight, obesity and the obesity-associated type 2 diabetes. Hyperinsulinemia, which is commonly seen as part of metabolic reprograming in the obese state, has been associated with higher risk of death and recurrence in BC. Up to 80% of breast cancers overexpress the insulin receptor (INSR) de novo, which correlates with worse prognosis. To directly probe the role of insulin signaling in mammary tumorigenesis in the mouse, we generated the MMTV-driven polyoma middle T (PyMT) and ErbB2/Her2 BC models, respectively, with coordinate mammary epithelium-restricted deletion of the INSR. In both models, deletion of either one or both copies of the INSR in the mammary gland led to a marked delay in tumor onset and burden, including in mice fed to mimic conditions of human obesity. Longitudinal monitoring of generated mouse models revealed that tumor initiation, rather than progression and metastasis, were impacted by INSR deletion. The similarity of phenotypes elicited by the deletion of one or both copies of INSR raises the possibility that there is a dose-dependent threshold for the contribution of INSR to mammary tumorigenesis.

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 burdens1% 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.

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

Metformin exhibits anticancer properties and is currently being explored as a therapeutic option for a variety of cancer types. Epidemiological studies demonstrate associations between metformin use in patients with type 2 diabetes and decreased cancer incidence and cancer-related mortality (Evans et al., 2005). Metformin also exhibits inhibitory effects on cancer cells in vitro and in mouse models (Anisimov et al., 2005; Zakikhani et al., 2006). Its mechanism of action is believed to involve both indirect (insulin-dependent) and direct (insulin-independent) effects.

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. Cancer Res; 70(20); 7776–87. ©2010 AACR.

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.

A recent convergence of clinical and epidemiological evidence suggests that hyperinsulinemia associated with obesity and type 2 diabetes leads to higher incidence and poor outcome of breast cancer (BC). To probe the role of insulin signaling in BC, we created the MMTV-driven polyoma middle T BC model mice with an inducible mammary epithelium-specific deletion of the insulin receptor (INSR). Deletion of INSR in the mammary gland considerably reduced the mammary tumour burden in the INSR knockout mice as compared to the INSR wildtype controls, without affecting the tumour onset. The average weight of individual tumours was similar across the genotypes, suggesting that deletion of INSR affected the tumour initiation potential of the epithelial cells rather than tumour growth. Deletion of INSR also affected the metastasis of mammary tumours to the lung, with the INSR knockout mice presenting with half the number of metastases compared to the INSR wildtype controls. Our ongoing efforts are focused on mapping the signaling events downstream of the INSR that govern tumour initiation and metastasis in our mice, as well as modeling the impact of obesity on tumors in this model system, with the ultimate goal of identifying the role of INSR signaling in BC development and progression. Citation Format: Yekaterina Poloz, Samar Mouaaz, Sonya Lam, Vuk Stambolic. Insulin receptor signaling contributes to mammary tumorigenesis in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 664.

Abstract 12 T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cell progenitors which affects both children and adults. Current therapies achieve cure in about 80% of pediatric patients, but only 40% of adults survive beyond 5 years. Disease relapse may be attributed to ineffective targeting of leukemia stem cells (LSCs) due to their presumed quiescence, resistance to apoptosis, enhanced expression of DNA repair enzymes and drug efflux pumps, and localization to protective or inaccessible niches. More efficient targeting of LSCs in this disease thus could offer great potential for improving patient outcomes. Recent interest in the role of reactive oxygen species (ROS) in cancer stem cell biology has been sparked by studies demonstrating that low ROS levels promote clonogenicity and radioresistance of breast cancer stem cells and that parthenolide specifically induces apoptosis of LSCs in AML and blast crisis CML by increasing levels of ROS in these cells. In T-ALL, activation of NOTCH1 signaling, either by mutation of NOTCH1 itself (50-60% of cases) or its negative regulator FBW7 (15% of cases), contributes to malignant behavior by blocking differentiation, promoting cellular proliferation/survival, and enhancing self-renewal. Similar gain-of-function Notch1 mutations are also observed to occur spontaneously in several murine T-ALL models at high frequency. To examine ROS levels in T-ALL LSCs, we first generated murine disease by retroviral transduction of bone marrow with a constitutively activated form of NOTCH1 termed “ΔE”. We utilized limiting dilution transplantation analyses of different cellular phenotypes isolated from primary ΔE leukemias to demonstrate that the CD44+ subset of CD4+ CD8+ (DP) cells were enriched for LSCs and exhibited lower ROS levels as compared to the CD44- DP subset and bulk cells. Since ROS levels were heterogeneous within the CD44+ DP subset, we combined these markers with DC-FDA staining for ROS and demonstrated as few as 100 ROSlow CD44+ DP cells are able to reproduce disease in 100% of recipients whereas 100,000 bulk cells are required to achieve the same effect. Because protein kinase C theta (PKCθ) was recently identified as an important mediator of ROS production in T-cells during activation-induced cell death and expression of PKCθ mRNA could be detected broadly in primary human and mouse T-ALL samples, we reasoned that this signaling intermediate might be a regulator of ROS levels in malignant T-ALL LSCs. In fact, using a combination of viral expression and knock-down approaches in both human and mouse T-ALLs as well as mouse PKCθnull ΔE leukemias, we demonstrate that PKCθ is a primary regulator ROS production in T-ALL. Moreover, low PKCθ/ROS levels correlate with resistance to chemotherapy and irradiation, and promote the competitive in vivo growth of T-ALL cells. Interestingly, we also find that Notch signaling represses PKCθ expression which in turn results in reduced ROS levels. Our results identify PKCθ as a novel target of Notch signaling in LSCs whose key malignant properties are highly dependent on low ROS levels. These findings provide mechanistic basis for rational design of LSC-targeted therapies in T-ALL. Disclosures: No relevant conflicts of interest to declare.