DNA methylation is a dynamic epigenetic mark that undergoes extensive changes during differentiation of self-renewing stem cells. However, whether these changes are the cause or consequence of stem cell fate remains unknown. Here, we show that alternative functional programs of hematopoietic stem cells (HSCs) are governed by gradual differences in methylation levels. Constitutive methylation is essential for HSC self-renewal but dispensable for homing, cell cycle control and suppression of apoptosis. Notably, HSCs from mice with reduced DNA methyltransferase 1 activity cannot suppress key myeloerythroid regulators and thus can differentiate into myeloerythroid, but not lymphoid, progeny. A similar methylation dosage effect controls stem cell function in leukemia. These data identify DNA methylation as an essential epigenetic mechanism to protect stem cells from premature activation of predominant differentiation programs and suggest that methylation dynamics determine stem cell functions in tissue homeostasis and cancer.

The transcription factor PU.1 occupies a central role in controlling myeloid and early B-cell development, and its correct lineage-specific expression is critical for the differentiation choice of hematopoietic progenitors. However, little is known of how this tissue-specific pattern is established. We previously identified an upstream regulatory cis element whose targeted deletion in mice decreases PU.1 expression and causes leukemia. We show here that the upstream regulatory cis element alone is insufficient to confer physiologic PU.1 expression in mice but requires the cooperation with other, previously unidentified elements. Using a combination of transgenic studies, global chromatin assays, and detailed molecular analyses we present evidence that PU.1 is regulated by a novel mechanism involving cross talk between different cis elements together with lineage-restricted autoregulation. In this model, PU.1 regulates its expression in B cells and macrophages by differentially associating with cell type-specific transcription factors at one of its cis-regulatory elements to establish differential activity patterns at other elements. (Blood. 2011;117(10):2827-2838)

A single-chain Fv (scFv) fragment derived from the murine antibody 4G7, specific for human lymphocyte CD19, was engineered for stability and expression in Escherichia coli in view of future use as a therapeutic protein. We compared two orthogonal knowledge-based procedures. In one approach, we designed a mutant with 14 single amino-acid substitutions predicted to correct destabilizing residues in the 4G7-wt sequence to create 4G7-mut. In the second variant, the murine CDRs were grafted to the human acceptor framework huVkappa3-huV(H)3, with 11 additional point mutations introduced to obtain a better match between CDR graft and acceptor framework, to arrive at 4G7-graft. Compared to 4G7-wt, 4G7-mut showed greater thermodynamic stability in guanidinium chloride-induced equilibrium denaturation experiments and somewhat greater stability in human serum. The loop graft maintained the comparatively high stability of the murine loop donor, but did not improve it further. Our analysis indicates that this is due to subtle strain introduced between CDRs and framework, mitigating the otherwise highly favorable properties of the human acceptor framework. This slight strain in the loop graft is also reflected in the binding affinities for CD19 on leukemic cells of 8.4 nM for 4G7-wt, 16.4 nM for 4G7-mut and 30.0 nM for 4G7-graft. This comparison of knowledge-based mutation and loop-grafting-based approaches will be important, when moving molecules forward to therapeutic applications.

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Introduction: Worldwide, breast cancer is the second most common cancer after lung cancer and the leading cause of cancer death in women. These facts underline the urgent need for improved therapeutic options. However, with marked progress in the understanding of breast cancer biology, it has become obvious that breast cancer is not a single disease but a collection of very heterogeneous entities both between patients as well as within a tumor. This variability poses a major challenge for translational research and the development of new drugs. Most preclinical models fail to reproduce this inter- and intra-tumor heterogeneity which might at least partly contribute to the unacceptably high attrition rates in clinical testing. Although traditionally used in vivo models such as cell line xenografts have been of great value in the past, with the advent of personalized health care, improved models are required to properly reflect the complex physiology. Aim and experimental procedure: To this end, we aim at the development and evaluation of orthotopic patient-derived xenograft (PDX) models for breast cancer. Such PDX models have been shown repeatedly to preserve some tumor heterogeneity and a genetic profile similar to the original primary tumor. Specifically, two different approaches are tested to transfer primary tumor material to orthotopic locations in immunodeficient mice. On the one hand, small tumor fragments are transplanted into the mammary fat pad (i.mfp) and secondly, intraductal injection is applied for tumor cell transfer. We hypothesize that the intraductal implantation of primary tumor cells mimics breast carcinogenesis more closely than existing xenografts. Consequently, such a model would provide a unique system to study the whole spectrum of tumor progression to invasive and metastatic disease. Results: So far, five subcutaneously established PDX models, which were used for proof-of-concept studies, successfully engrafted using i.mfp transfer. As expected, histological analysis showed that tumor characteristics, such as expression of Her2, were faithfully retained in the grafted tumors. Intriguingly, two of the established PDX models tested were successfully injected intraductally and yielded invasive mammary carcinomas. These, to our knowledge, are the first breast cancer PDX models shown to grow invasively upon intraductal transfer. Furthermore, preliminary results showed an enhanced penetration of the tumor by lymphatic vessels after orthotopic transfer, especially after intraductal injection, compared to ectopic transplantation (subcutaneous), suggesting a more physiological growth and vessel supply. On top, we have observed tumor engraftment and invasive growth of seven primary samples (patient surgical specimen) upon i.mfp transfer, one of which also developed from intraductal injection, confirming that invasive tumors can form upon direct injection of patient material into milks ducts of mice. Conclusion and Outlook: Taken together, we want to create novel preclinical models for breast cancer which are able to provide a good phenocopy of several subtypes of human tumors and can consequently serve as models with improved predictivity. First results indicate that orthotopic transfer of breast PDX models displays a very physiological modeling of breast cancer and, importantly, that the most orthotopic placement of tumor cells (intra-ductal injection) allows invasive growth of breast tumor cells. In the future, we plan to include further models for intraductal injection to allow a thorough comparison between the two techniques and a reciprocal comparison to the original tumor. Additionally, application of the two implantation techniques in an immune-system humanized mouse background could provide very complex and physiological preclinical tools for cancer immunotherapeutic drug candidates. Citation Format: Lena Vockentanz, Adam Nopora. Development of next-generation breast cancer PDX models by applying intra mammary fat pad and intraductal tumor transfer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B28.