Mast cell deficiency prevents BCR::ABL1 induced splenomegaly and cytokine elevation in a CML mouse model.

The persistence of leukemic stem cells (LSCs) represents a problem in the therapy of chronic myeloid leukemia (CML). Hence, it is of utmost importance to explore the underlying mechanisms to develop new therapeutic approaches to cure CML. Using the genetically engineered ScltTA/TRE-BCR::ABL1 mouse model for chronic phase CML, we previously demonstrated that the loss of the docking protein GAB2 counteracts the infiltration of mast cells (MCs) in the bone marrow (BM) of BCR::ABL1 positive mice. Here, we show for the first time that BCR::ABL1 drives the cytokine independent expansion of BM derived MCs and sensitizes them for FcεRI triggered degranulation. Importantly, we demonstrate that genetic mast cell deficiency conferred by the Cpa3Cre allele prevents BCR::ABL1 induced splenomegaly and impairs the production of pro-inflammatory cytokines. Furthermore, we show in CML patients that splenomegaly is associated with high BM MC counts and that upregulation of pro-inflammatory cytokines in patient serum samples correlates with tryptase levels. Finally, MC-associated transcripts were elevated in human CML BM samples. Thus, our study identifies MCs as essential contributors to disease progression and suggests considering them as an additional target in CML therapy. Mast cells play a key role in the pro-inflammatory tumor microenvironment of the bone marrow. Shown is a cartoon summarizing our results from the mouse model. BCR::ABL1 transformed MCs, as part of the malignant clone, are essential for the elevation of pro-inflammatory cytokines, known to be important in disease initiation and progression.

Oncogenic KrasG12D Activation in the Nonhematopoietic Bone Marrow Microenvironment Causes Myelodysplastic Syndrome in Mice.

The bone marrow microenvironment (BMME) is key player in regulation and maintenance of hematopoiesis. Oncogenic RAS mutations, causing constitutive activation of multiple tumor-promoting pathways, are frequently found in human cancer. So far in hematologic malignancies, RAS mutations have only been reported to occur in hematopoietic cells. In this study, we investigated the effect of oncogenic Kras expression in the BMME in a chimeric mouse model. We observed that an activating mutation of Kras in the nonhematopoietic system leads to a phenotype resembling myelodysplastic syndrome (MDS) characterized by peripheral cytopenia, marked dysplasia within the myeloid lineage as well as impaired proliferation and differentiation capacity of hematopoietic stem and progenitor cells. The phenotypic changes could be reverted when the BM was re-isolated and transferred into healthy recipients, indicating that the KrasG12D -activation in the nonhematopoietic BMME was essential for the MDS phenotype. Gene expression analysis of sorted nonhematopoietic BM niche cells from KrasG12D mice revealed upregulation of multiple inflammation-related genes including IL1-superfamily members (Il1α, Il1ß, Il1f9) and the NLPR3 inflammasome. Thus, pro-inflammatory IL1-signaling in the BMME may contribute to MDS development. Our findings show that a single genetic change in the nonhematopoietic BMME can cause an MDS phenotype. Oncogenic Kras activation leads to pro-inflammatory signaling in the BMME which impairs HSPCs function. IMPLICATIONS: These findings may help to identify new therapeutic targets for MDS.

B-Raf deficiency impairs tumor initiation and progression in a murine breast cancer model.

Copy number gains, point mutations and epigenetic silencing events are increasingly observed in genes encoding elements of the Ras/Raf/MEK/ERK signaling axis in human breast cancer. The three Raf kinases A-Raf, B-Raf, and Raf-1 have an important role as gatekeepers in ERK pathway activation and are often dysregulated by somatic alterations of their genes or by the aberrant activity of receptor tyrosine kinases (RTKs) and Ras-GTPases. B-Raf represents the most potent Raf isoform and a critical effector downstream of RTKs and RAS proteins. Aberrant RTK signaling is mimicked by the polyoma middle T antigen (PyMT), which activates various oncogenic signaling pathways, incl. the RAS/ERK axis, in a similar manner as RTKs in human breast cancer. Mammary epithelial cell directed expression of PyMT in mice by the MMTV-PyMT transgene induces mammary hyperplasia progressing over adenoma to metastatic breast cancer with an almost complete penetrance. To understand the functional role of B-Raf in this model for luminal type B breast cancer, we crossed MMTV-PyMT mice with animals that either lack B-Raf expression in the mammary gland or express the signaling impaired B-RafAVKA mutant. The AVKA mutation prevents phosphorylation of T599 and S602 in the B-Raf activation loop and thereby activation of the kinase by upstream signals. We demonstrate for the first time that B-Raf expression and activation is important for tumor initiation in vivo as well as for lung metastasis. Isogenic tumor cell lines generated from conditional Braf knock-out or knock-in mice displayed a reduction in EGF-induced ERK pathway activity as well as in proliferation and invasive growth in three-dimensional matrigel cultures. Our results suggest that B-Raf, which has been hardly studied in the context of breast cancer, represents a critical effector of the PyMT oncoprotein and invite for an assessment of its functional role in human breast cancer.

The Atypical Kinase RIOK1 Promotes Tumor Growth and Invasive Behavior.

Despite being overexpressed in different tumor entities, RIO kinases are hardly characterized in mammalian cells. We investigated the role of these atypical kinases in different cancer cells. Using isogenic colon-, breast- and lung cancer cell lines, we demonstrate that knockdown of RIOK1, but not of RIOK2 or RIOK3, strongly impairs proliferation and invasiveness in conventional and 3D culture systems. Interestingly, these effects were mainly observed in RAS mutant cancer cells. In contrast, growth of RAS wildtype Caco-2 and Bcr-Abl-driven K562 cells is not affected by RIOK1 knockdown, suggesting a specific requirement for RIOK1 in the context of oncogenic RAS signaling. Furthermore, we show that RIOK1 activates NF-κB signaling and promotes cell cycle progression. Using proteomics, we identified the pro-invasive proteins Metadherin and Stathmin1 to be regulated by RIOK1. Additionally, we demonstrate that RIOK1 promotes lung colonization in vivo and that RIOK1 is overexpressed in different subtypes of human lung- and breast cancer. Altogether, our data suggest RIOK1 as a potential therapeutic target, especially in RAS-driven cancers.