Tumor necrosis factor restricts hematopoietic stem cell activity in mice: involvement of two distinct receptors.

Whereas maintenance of hematopoietic stem cells (HSCs) is a requisite for life, uncontrolled expansion of HSCs might enhance the propensity for leukemic transformation. Accordingly, HSC numbers are tightly regulated. The identification of physical cellular HSC niches has underscored the importance of extrinsic regulators of HSC homeostasis. However, whereas extrinsic positive regulators of HSCs have been identified, opposing extrinsic repressors of HSC expansion in vivo have yet to be described. Like many other acute and chronic inflammatory diseases, bone marrow (BM) failure syndromes are associated with tumor necrosis factor-α (TNF) overexpression. However, the in vivo relevance of TNF in the regulation of HSCs has remained unclear. Of considerable relevance for normal hematopoiesis and in particular BM failure syndromes, we herein demonstrate that TNF is a cell-extrinsic and potent endogenous suppressor of normal HSC activity in vivo in mice. These effects of TNF involve two distinct TNF receptors

A Novel Role for CCL3 (MIP-1α) in Myeloma-induced Bone Disease via Osteocalcin Downregulation and Inhibition of Osteoblast Function

Upregulation of cytokines and chemokines is a frequent finding in multiple myeloma (MM). CCL3 (also known as MIP-1α) is a pro-inflammatory chemokine whose levels in the MM microenvironment correlate with osteolytic lesions and tumor burden. CCL3 and its receptors, CCR1 and CCR5, contribute to the development of bone disease in MM by supporting tumor growth and regulating osteoclast (OC) differentiation. Here, we identify inhibition of osteoblast (OB) function as an additional pathogenic mechanism in CCL3-induced bone disease. MM-derived and exogenous CCL3 represses mineralization and osteocalcin production by primary human bone marrow stromal cells and HS27A cells. Our results suggest that CCL3 effects on OBs are mediated by ERK activation and subsequent downregulation of the osteogenic transcription factor osterix. CCR1 inhibition reduced ERK phosphorylation and restored both osterix and osteocalcin expression in the presence of CCL3. Finally, treating SCID-hu mice with a small molecule CCR1 inhibitor suggests an upregulation of osteocalcin expression along with OC downregulation. Our results show that CCL3, in addition to its known catabolic activity, reduces bone formation by inhibiting OB function and therefore contributes to OB/OC uncoupling in MM

The FLT3 ligand potently and directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells in vitro: synergistic interactions with interleukin (IL) 11, IL-12, and other hematopoietic growth factors

The recently cloned murine flt3 ligand (FL) was studied for its ability to stimulate the growth of primitive (Lin-Sca-1+) and more committed (Lin-Sca-1-) murine bone marrow progenitor cells, alone and in combination with other hematopoietic growth factors (HGFs). Whereas FL was a weak proliferative stimulator alone, it potently synergized with a number of other HGFs, including all four colony-stimulating factor (CSF), interleukin (IL) 6, IL-11, IL-12, and stem cell factor (SCF), to promote the colony formation of Lin-Sca-1+, but not Lin-Sca-1- or erythroid progenitor cells. The synergistic activity of FL was concentration dependent, with maximum stimulation occurring at 250 ng/ml, and was observed when cells were plated at a concentration of one cell per culture, suggesting that its effects are directly mediated. 2 wk of treatment with FL in combination with IL-3 or SCF resulted in the production of a high proportion of mature myeloid cells (granulocytes and macrophages), whereas the combination of FL with G- CSF, IL-11, or IL-12 resulted predominantly in the formation of cells with an immature blast cell appearance. Accordingly, FL in combination with G-CSF or IL-11 expanded the number of progenitors more than 40- fold after 2 wk incubation. Thus, FL emerges as a potent synergistic HGF, that in combination with numerous other HGFs, can directly stimulate the proliferation, myeloid differentiation, and expansion of primitive hematopoietic progenitor cells

Phospho-histone H3 and MMAE levels in HEK293-GCC and PHTX-09c xenograft tumors following TAK-264 treatment.

<p>(A) HEK293-GCC and (B) PHTX-09 xenograft tumors were harvested at the indicated times post-treatment, with three animals per time point. Control tumors were taken from three untreated mice. The average percentage of phospho-histone H3-positive cells and average level of MMAE tumor concentration are plotted, with error represented as standard deviation. GCC, guanylyl cyclase C; MMAE, monomethyl auristatin E.</p

A monomethyl auristatin E-conjugated antibody to guanylyl cyclase C is cytotoxic to target-expressing cells <i>in vitro</i> and <i>in vivo</i>

<div><p>Guanylyl cyclase C (GCC) is a cell-surface protein that is expressed by normal intestinal epithelial cells, more than 95% of metastatic colorectal cancers (mCRC), and the majority of gastric and pancreatic cancers. Due to strict apical localization, systemically delivered GCC-targeting agents should not reach GCC in normal intestinal tissue, while accessing antigen in tumor. We generated an investigational antibody-drug conjugate (TAK-264, formerly MLN0264) comprising a fully human anti-GCC monoclonal antibody conjugated to monomethyl auristatin E via a protease-cleavable peptide linker. TAK-264 specifically bound, was internalized by, and killed GCC-expressing cells <i>in vitro</i> in an antigen-density-dependent manner. In GCC-expressing xenograft models with similar GCC expression levels/patterns observed in human mCRC samples, TAK-264 induced cell death, leading to tumor regressions and long-term tumor growth inhibition. TAK-264 antitumor activity was generally antigen-density-dependent, although some GCC-expressing tumors were refractory to TAK-264-targeted high local concentrations of payload. These data support further evaluation of TAK-264 in the treatment of GCC-expressing tumors.</p></div

TAK-264 induces tumor regression and long-term tumor growth delay of GCC-expressing tumors.

<p>Female SCID mice bearing (A) GCC-expressing HEK293 xenograft tumors and (B) PHTX-09c tumors, a patient-derived xenograft model of mCRC, were treated when the tumor reached approximately 200 mm³ with either vehicle, non-targeting control antibody 209-vcMMAE, free MMAE, or TAK-264 at various doses on a weekly dosing schedule indicated by the black triangles. Average tumor volume was determined at multiple time points following start of treatment and is shown ± S.E.M.</p

Characterization of TAK-264 binding, internalization, and <i>in vitro</i> cytotoxicity.

<p>(A) Unconjugated anti-GCC monoclonal antibody and TAK-264 bind to GCC-expressing cells. Flow cytometry was performed with unconjugated anti-GCC monoclonal antibody and TAK-264 on HEK293-GCC or HEK293-vector cells. Mean fluorescence intensity is plotted versus monoclonal antibody concentration ranging from 0.001–1 μg/mL. (B) TAK-264 is internalized by GCC-expressing cells. Internalization assays were performed with TAK-264 on HEK-GCC cells. Antibodies were incubated with cells at (i) 4°C for 30 min or at (ii) 37°C for 3 h, prior to fixing and staining with secondary antibody. Immunofluorescence was visualized with a confocal microscope (Zeiss Pascal) and analyzed using Axiovert software. (C) Time-dependent accumulation of MMAE in samples incubated with 1 μg TAK-264 at 37°C, 5% CO₂, and analyzed by LC/MS/MS. (D) <i>In vitro</i> cytotoxicity of TAK-264. Cell viability was assessed 4 days after incubation of HEK293-GCC cells or HEK293-vector cells with increasing concentrations of unconjugated and MMAE-conjugated anti-GCC monoclonal antibody. The average of three independent experiments is plotted with error represented as SEM. GCC, guanylyl cyclase C; LC/MS/MS, liquid chromatography–tandem mass spectrometry; MMAE, monomethyl auristatin E; SEM, standard error of the mean.</p