The most common metastatic lesions of prostate cancer are in bone and can be classified into three distinct pathology subtypes: lytic, blastic, and an indeterminate mixture of both. We investigated a cohort of decalcified formalin-fixed and paraffin-embedded (FFPE) patient specimens from the bone that contained metastatic prostate cancer with lytic or blastic features. These tissue sections were utilized for immunohistochemistry (IHC) staining, isolation of RNA for gene expression, and Digital Spatial Profiling (DSP) of changes in both the tumor and microenvironment. A diverse set of unique immune cell populations and signaling pathways to both lytic and blastic types of prostate cancer metastases were present. In blastic lesions immune cells were enriched for pSTAT3 and components of the JAK-STAT pathway. In lytic-type lesions, immune cells were enriched for pAKT activity and components of the PI3K-AKT pathway. Enrichment for immune checkpoints including PD-L1, B7-H4, OX40L, and IDO-1 were identified in blastic prostate cancer, providing new therapeutic targets for patients with bone metastases. Biopsies could guide selection of patients into appropriate therapeutic interventions based on protein levels and RNA expression of desired targets in metastatic disease. Molecular pathology has been an excellent complement to the diagnosis, treatment, and management of primary tumors and could be successfully extended to patients with metastatic lesions.

Cell lineage specification is a tightly regulated process that is dependent on appropriate expression of lineage and developmental stage-specific transcriptional programs. Here, we show that Chromodomain Helicase DNA-binding protein 4 (CHD4), a major ATPase/helicase subunit of Nucleosome Remodeling and Deacetylase Complexes (NuRD) in lymphocytes, is essential for specification of the early B cell lineage transcriptional program. In the absence of CHD4 in B cell progenitors in vivo, development of these cells is arrested at an early pro-B-like stage that is unresponsive to IL-7 receptor signaling and unable to efficiently complete V(D)J rearrangements at Igh loci. Our studies confirm that chromatin accessibility and transcription of thousands of gene loci are controlled dynamically by CHD4 during early B cell development. Strikingly, CHD4-deficient pro-B cells express transcripts of many non-B cell lineage genes, including genes that are characteristic of other hematopoietic lineages, neuronal cells, and the CNS, lung, pancreas, and other cell types. We conclude that CHD4 inhibits inappropriate transcription in pro-B cells. Together, our data demonstrate the importance of CHD4 in establishing and maintaining an appropriate transcriptome in early B lymphopoiesis via chromatin accessibility.

Summary In the absence of EBF1, B cell development is arrested at an uncommitted progenitor stage that exhibits increased lineage potentials. Previously, we investigated the roles of EBF1 (E) and its DNA binding partner Runx1 (R) by evaluating B lymphopoiesis in single (Ehet and Rhet) and compound haploinsufficent (ERhet) mice. Here, we demonstrate that reduced Ebf1 gene dosage results in the inappropriate expression of NK cell lineage-specific genes in B cell progenitors. Moreover, prolonged expression of Ly6a/Sca-1 suggested the maintenance of a relatively undifferentiated phenotype. These effects were exacerbated by reduced expression of Runx1 and occurred despite expression of Pax5. Repression of inappropriately expressed genes was restored in most pre-B and all immature B cells of ERhet mice. Enforced EBF1 expression repressed promiscuous transcription in pro-B cells of ERhet mice and in Ebf1−/−Pax5−/− fetal liver cells. Together, our studies suggest that normal levels of EBF1 are critical for maintaining B cell identity by directing repression of non-B cell-specific genes.

The Bone Morphogenetic Protein (BMP) pathway is a member of the TGFβ signaling family and has complex roles in cancer. BMP signaling is rarely mutated and can be frequently overexpressed in many human cancers. The dichotomous role of BMPs as both tumor promoters and suppressors appears to be largely context based in both the cancer cell and the surrounding microenvironment. Myeloid cells including macrophages and neutrophils have been shown to be tumor promoting when stimulated from BMPs. We found that conditional deletion of BMPR1a in myeloid cells (LysMCre) restricts tumor progression in a syngeneic mouse prostate cancer model. Specific changes occurred in myeloid cells both in tumor bearing mice and tumor naïve mice throughout multiple tissues. We profiled myeloid subsets in the bone marrow, spleen and primary tumor and found myeloid BMPR1a loss altered the differentiation and lineage capability of distinct populations by histologic, flow cytometry and high dimensional mass cytometry analysis. We further confirmed the requirement for BMP signaling with pharmacologic inhibition of THP-1 and Raw264.7 activated into M2 macrophages with the BMP inhibitor DMH1. M2 polarized primary bone marrow derived cells from LysMCre BMPR1a knockout mice indicated a distinct requirement for BMP signaling in myeloid cells during M2 activation. These results indicate a unique necessity for BMP signaling in myeloid cells during tumor progression.

From the 33,000 men in the U.S. who die from prostate cancer each year, the majority of these patients exhibit metastatic disease with bone being the most common site of metastasis. Prostate cancer bone metastases are commonly blastic, exhibiting new growth of unhealthy sclerotic bone, which can cause painful skeletal related events. Patient’s current care entails androgen deprivation therapy, anti-resorptive agents, radiation, and chemotherapy to help control the spread of the cancer but little intervention is available to treat blastic bone disease. The transforming growth factor beta (TGFβ) and bone morphogenetic protein (BMP) pathways are known to regulate bone growth and resorption of destructive lytic bone lesions, yet the role of TGFβ/BMP signaling in prostate cancer blastic vs lytic bone lesions are not fully understood. We hypothesized that to target the BMP/TGFβ pathway, a useful biomarker of bone lytic or blastic pathology would have superior response. We show distinct BMP vs. TGFβ signaling in clinical samples of human prostate cancer bone metastases with either lytic or blastic pathologies. BMPs exhibit distinct effects on bone homeostasis, so to examine the effect of BMP inhibition on healthy bone, we treated mice with the BMP receptor small molecule antagonist DMH1 and saw a modest temporary improvement in bone health, with increased trabecular bone. We next sought to use the BMP inhibitor DMH1 to treat bone metastasis engraftment seeded by a caudal artery injection of the lytic human prostate cell line PC3 in immunodeficient mice. The colonization by PC3 cells to the bone were restricted with DMH1 treatment and bone health was importantly preserved. We next proceeded to test BMP inhibition in an injury model of established bone metastasis via intratibial injection of the MYC-CaP mouse prostate cell line into FVBN syngeneic mice. DMH1 treated mice had a modest decrease in trabecular bone and reduced lymphocytes in circulation without affecting tumor growth. Taken together we show unique responses to BMP inhibition in metastatic prostate cancer in the bone. These studies suggest that profiling bone lesions in metastatic prostate cancer can help identify therapeutic targets that not only treat the metastatic tumor but also address the need to better treat the distinct tumor induced bone disease.