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K. Rajewsky Lab

Immune Regulation and Cancer


Human B cell malignancies represent a major medical problem because of their abundance and limitations of available therapies.

We study B cell lymphomagenesis in comparison to normal B cell physiology, through genetic mouse models generated by conditional gene targeting.

Recent achievements include the identification of antagonizing PI3 Kinase and FOXO1 activities as key elements of germinal center B cell differentiation, and their cooperativity in B cell lymphomagenesis. Additional new insights relate to the mechanism of immune surveillance of Epstein-Barr-Virus (EBV) infected and transformed B cells, as well as various aspects of normal B cell development including its control by miRNAs.

A major new direction of our work comes from the availability of CRISPR/Cas-mediated targeted mutagenesis.We have improved this tool to introduce tailored mutations and insertions, and used it to generate genetically modified mice by zygote mutagenesis. A mouse strain allowing conditional expression of the Cas9 endonuclease is being used for genetic screens in primary cells, and we have added gene editing in human cells to our agenda.



The interplay between B cell antigen receptor and Toll-like receptors in the B cell proliferative response
(In the B cell proliferative response K. Otipoby*, A. Waisman*, E. Derudder, L. Srinivasan*, A. Franklin  *Former group members)
We had earlier shown that the B cell antigen receptor (BCR) is a key survival determi
nant for mature B cells, depending on signals of the phosphoinositide 3-kinase (PI3K) pathway. We now demonstrated that when BCR-deficient B cells are kept alive by a Bcl2 transgene, they fail to initiate a proliferative response to Toll-like receptor (TLR) engagement (Otipoby et al. 2015). As in the case of BCR-dependent B cell survival, the proliferative response of the BCR-deficient cells could be rescued by PI3K signaling. Altogether a model emerges in which B cells integrate signals from the BCR (recognizing specific antigenic determinants) and co-receptors (responding to T cell help or pathogen-associated molecular patterns) in the control of their proliferative response. In Myc-transformed mouse B cells the BCR controls the competitive fitness of the tumor cells (collaboration with S. Casola, IFOM, Milan).

The germinal center reaction and B cell lymphomagenesis
(S. Sander, V.T. Chu, T. Yasuda, A. Franklin, R. Graf, D. Calado*, K. Imami+, M. Selbach+, M. Di Virgilio+, L. Bullinger+, E. Kabrani  *Former group members + Collaborators)
In T cell-dependent immune responses antigen-specific B cells undergo successive rounds of proliferation and selection in histological structures called germinal centers (GCs). Proliferation is accompanied by somatic hypermutation (SHM) and class-switching (CSR) of the antibodies expressed by the cells, and cells expressing high-affinity antibodies are selected into the pool of memory B cells and long-lived plasma cells. Because of their extensive proliferation accompanied by DNA breaks introduced by SHM and CSR, GC B cells are at the origin of most B cell lymphomas. We had earlier modeled a typical GC-derived malignancy, Burkitt lymphoma, in mice, by targeted induction of c-MYC and PI3K activity in GCs (Sander et al. Cancer Cell 2012). The resulting lymphomas closely resembled human Burkitt, including tertiary mutations selected during tumor progression. Among the latter we discovered recurrent activating mutations of the FOXO1 transcription factor. As this resulted in co-expression of FOXO1 and PI3K signaling, mutually exclusive in normal physiology where PI3K inactivates FOXO1, we investigated the role of FOXO1 and PI3K signaling in the GC reaction. We found a clear division of labor, with PI3K activity limited to the so-called GC Light Zone (LZ), where mutant B cells are selected, and FOXO1 nuclear expression largely restricted to the Dark Zone (DZ), where GC B cells proliferate and undergo SHM. FOXO1 turned out to be a master regulator of the GC DZ, controlling more than 60% of DZ-specific genes; in its absence, GC DZs were not formed (Fig. 1B), and, although B cell proliferation and SHM were unaffected, cellular selection and CSR in GCs were profoundly disturbed. We concluded that proper sequential proliferation/mutation and selection of GC cells is controlled by the PI3K-FOXO1 antagonism and essential for efficient antibody affinity maturation in GCs (Sander et al. 2015). Interestingly, the Burkitt-like lymphoma cells carrying activating FOXO1 mutations are dependent on concomitant PI3K and FOXO1 activity, as shown by CRISPR/Cas9 mediated targeted mutagenesis (unpublished data). Present studies explore the mechanism of this unusual cooperation and its role in lymphoma pathogenesis.

Mouse models of Diffuse Large B Cell Lymphoma and Multiple Myeloma

(B. Zhang*, D. Calado*, Z. Wang+, S. Fröhler+, K. Köchert*, Y. Qian+, S. Koralov*, M. Schmidt-Supprian*, Y. Sasaki*, C. Unitt+, S. Rodig+, W. Chen+, R. Dalla-Favera+, F.W. Alt+, L. Pasqualucci+, K. Schmidt, U. Sack*, W. Winkler, M. Janz)
Various attempts have been and are being made to model DLBCL and MM in mice. In a recent study we showed that a fraction of human DLBCLs exhibit activation of the alternative NF-kB pathway, and that activation of this pathway plays indeed an oncogenic role in a mouse model of DLBCL (Zhang et al. 2015). This complements previous evidence that activation of the canonical NF-kB pathway, together with other oncogenic events, drives DLBCL of the ABC subtype. Ongoing work explores the role of other recurrent mutations in DLBCL in tumor pathogenesis, such as activating mutations in signaling pathways emanating from the BCR and TLRs. We are also developing mouse models of MM and other tumor entities through sequential activation of oncogenic events recurrently identified in the human.

Epstein-Barr-Virus driven pathologies and immune surveillance; Hodgkin lymphoma (collaboration with the Janz/Mathas/Dörken group)
(T. Yasuda, T. Sommermann, T. Weber, T. Wirtz, S. Li, M. Janz)
Since our discovery many years ago that the tumor cells in Hodgkin Lymphoma (HL), the so-called Hodgkin & Reed-Sternberg (HRS) cells, originate from proapoptotic GC B cells which, in EBV+ cases, have been rescued by expression of the EBV proteins LMP1 and LMP2A, we develop mouse models of EBV infection and the immune surveillance of EBV-infected B cells, as well as EBV pathologies such as X-linked Lymphoproliferative Syndrome (XLP), Post-Transplant Immunoproliferative Disorder (PTLD), and EBV+ B cell lymphomas including HL. Human B cells infected and transformed by EBV rapidly expand, but are efficiently eliminated by T and NK cells, leaving behind a small pool of latently infected cells. When immune surveillance is compromised, the EBV infected cells cannot be controlled and lethal pathologies ensue. In earlier work we described that both EBV immune surveillance and pathologies can be modeled in mice by B cell-specific expression of the EBV proteins LMP1 and -2A (Zhang et al. Cell 2012; Yasuda et al. Cold Spring Harb Symp Quant Biol 2013). Fig. 2B-D shows our conditional,
Cre recombinase dependent LMP alleles and the expansion of LMP+ B cells and tumor formation in the absence of immune surveillance. Ongoing work explores the immunogenicity and T-cell recognition of LMP+ B cells and tumors, as well as the molecular mechanisms by which LMP+ B cells develop into monoclonal lymphomas in vivo. We have also developed mouse models of acute EBV infection and a monogenic inherited fatal EBV-driven lymphoproliferative human disease. With respect to HL we refer the reader to the report of M. Janz and colleagues.

Gene editing in somatic cells and mouse zygotes
(V.T. Chu, T. Weber, R. Graf, S. Sander, T. Sommermann, R. Kühn)
Realizing the profound impact of the CRISPR/Cas9 revolution on our work, we have, in collaboration with R. Kühn, attempted to enhance homology-directed DNA repair (HDR) at the expense of non-homologous end joining (NHEJ) in CRISPR/Cas9-mediated mutagenesis, in order to optimize this approach towards the introduction of precise modifications into genomic sequences. Chemical or genetic inhibition of the NHEJ pathway indeed led to a strong increase of HDR and almost complete suppression of NHEJ. We also succeeded in the rapid generation of mice carrying knock-out or conditional alleles through CRISPR/Cas9 mutagenesis of C57BL/6 zygotes (Fig. 3B). Particularly useful for our future work is a strain carrying a conditional Cas9 allele. We have shown that this strain allows efficient targeted mutagenesis and genetic screens in primary cells through transfection of guide-RNAs (Chu, Graf et al. 2016). We are in the process of extending our work to gene editing in human cells.

Other work
(MDC lab members involved: E. Derudder, A. Franklin, R. Graf, V. Labi, T. Yasuda)
Due to space limitations, we refer the reader to the literature and only mention a few projects, which are close to completion. These relate to NF-kB signaling and BCR specificity in B cell homeostasis and B1/B2 subset determination, the role and functional impact of Tet enzymes in lineage-specific DNA demethylation (with Y. Bergmann and H. Ceder, Hebrew University), and an unexpected, vital role of miRNA seed matches in the ubiquitously expressed proapoptotic Bim gene (with C. Birchmeier, M. Landthaler and N. Rajewsky). An AID-related project was initiated by A. Franklin with O. Daumke and M. Di Virgilio.