RAG-Mediated DNA Breaks Attenuate PU.1 Activity in Early B Cells through Activation of a SPIC-BCLAF1 Complex.

Early B cell development is regulated by stage-specific transcription factors. PU.1, an ETS-family transcription factor, is essential for coordination of early B cell maturation and immunoglobulin gene (Ig) rearrangement. Here we show that RAG DNA double-strand breaks (DSBs) generated during Ig light chain gene (Igl) rearrangement in pre-B cells induce global changes in PU.1 chromatin binding. RAG DSBs activate a SPIC/BCLAF1 transcription factor complex that displaces PU.1 throughout the genome and regulates broad transcriptional changes. SPIC recruits BCLAF1 to gene-regulatory elements that control expression of key B cell developmental genes. The SPIC/BCLAF1 complex suppresses expression of the SYK tyrosine kinase and enforces the transition from large to small pre-B cells. These studies reveal that RAG DSBs direct genome-wide changes in ETS transcription factor activity to promote early B cell development.

Ex Vivo and In Vivo Evaluation of Overexpressed VLA-4 in Multiple Myeloma Using LLP2A Imaging Agents.

UNLABELLED: Very-late-antigen-4 (VLA-4, α4ß1 integrin, CD49d/CD29) is a transmembrane adhesion receptor that plays an important role in cancer and immune responses. Enhanced VLA-4 expression has been observed in multiple myeloma (MM) cells and surrounding stroma. VLA-4 conformational activation has been associated with MM pathogenesis. VLA-4 is a promising MM imaging and therapeutic biomarker. METHODS: Specificity of (64)Cu-LLP2A ((64)Cu-CB-TE1A1P-PEG4-LLP2A), a high-affinity VLA-4 peptidomimetic-based radiopharmaceutical, was evaluated in α4 knock-out mice and by competitive blocking in wild-type tumor-bearing mice. (64)Cu-LLP2A PET/CT (static and dynamic) imaging was conducted in C57BL6/KaLwRij mice bearing murine 5TGM1-GFP syngeneic tumors generated after intravenous injection via the tail. Blood samples were collected for serum protein electrophoresis. Bone marrow and splenic cells extracted from tumor-bearing and control mice (n= 3/group) were coincubated with the optical analog LLP2A-Cy5 and mouse B220, CD4, Gr1, and Mac1 antibodies and analyzed by fluorescence-activated cell sorting. Human radiation dose estimates for (64)Cu-LLP2A were extrapolated from mouse biodistribution data (6 time points, 0.78 MBq/animal, n= 4/group). Ten formalin-fixed paraffin-embedded bone marrow samples from deceased MM patients were stained with LLP2A-Cy5. RESULTS: (64)Cu-LLP2A and LLP2A-Cy5 demonstrated high specificity for VLA-4-positive mouse 5TGM1-GFP myeloma and nonmalignant inflammatory host cells such as T cells and myeloid/monocytic cells. Ex vivo flow cytometric analysis supported a direct effect of myeloma on increased VLA-4 expression in host hematopoietic microenvironmental elements. SUVs and the number of medullar lesions detected by (64)Cu-LLP2A PET corresponded with increased monoclonal (M) protein (g/dL) in tumor-bearing mice over time (3.29 ± 0.58 at week 0 and 9.97 ± 1.52 at week 3). Dynamic PET with (64)Cu-LLP2A and (18)F-FDG demonstrated comparable SUV in the prominent lesions in the femur. Human radiation dose estimates indicated urinary bladder wall as the dose-limiting organ (0.200 mGy/MBq), whereas the dose to the red marrow was 0.006 mGy/MBq. The effective dose was estimated to be 0.017 mSv/MBq. Seven of the ten human samples displayed a high proportion of cells intensely labeled with LLP2A-Cy5 probe. CONCLUSION: (64)Cu-LLP2A and LLP2A-Cy5 demonstrated binding specificity for VLA-4 in an immune-competent murine MM model. (64)Cu-LLP2A displayed favorable dosimetry for human studies and is a potential imaging candidate for overexpressed VLA-4.

Small Molecule MYC Inhibitor Conjugated to Integrin-Targeted Nanoparticles Extends Survival in a Mouse Model of Disseminated Multiple Myeloma.

Multiple myeloma pathogenesis is driven by the MYC oncoprotein, its dimerization with MAX, and the binding of this heterodimer to E-Boxes in the vicinity of target genes. The systemic utility of potent small molecule inhibitors of MYC-MAX dimerization was limited by poor bioavailability, rapid metabolism, and inadequate target site penetration. We hypothesized that new lipid-based MYC-MAX dimerization inhibitor prodrugs delivered via integrin-targeted nanoparticles (NP) would overcome prior shortcomings of MYC inhibitor approaches and prolong survival in a mouse model of cancer. An Sn 2 lipase-labile prodrug inhibitor of MYC-MAX dimerization (MI1-PD) was developed which decreased cell proliferation and induced apoptosis in cultured multiple myeloma cell lines alone (P < 0.05) and when incorporated into integrin-targeted lipid-encapsulated NPs (P < 0.05). Binding and efficacy of NPs closely correlated with integrin expression of the target multiple myeloma cells. Using a KaLwRij metastatic multiple myeloma mouse model, VLA-4-targeted NPs (20 nm and 200 nm) incorporating MI1-PD (D) NPs conferred significant survival benefits compared with respective NP controls, targeted (T) no-drug (ND), and untargeted (NT) control NPs (T/D 200: 46 days vs. NT/ND: 28 days, P < 0.05 and T/D 20: 52 days vs. NT/ND: 29 days, P = 0.001). The smaller particles performed better of the two sizes. Neither MI1 nor MI1-PD provided survival benefit when administered systemically as free compounds. These results demonstrate for the first time that a small molecule inhibitor of the MYC transcription factor can be an effective anticancer agent when delivered using a targeted nanotherapy approach.

Very late antigen-4 (α(4)ß(1) Integrin) targeted PET imaging of multiple myeloma.

Biomedical imaging techniques such as skeletal survey and (18)F-fluorodeoxyglucose (FDG)/Positron Emission Tomography (PET) are frequently used to diagnose and stage multiple myeloma (MM) patients. However, skeletal survey has limited sensitivity as it can detect osteolytic lesions only after 30-50% cortical bone destruction, and FDG is a marker of cell metabolism that has limited sensitivity for intramedullary lesions in MM. Targeted, and non-invasive novel probes are needed to sensitively and selectively image the unique molecular signatures and cellular processes associated with MM. Very late antigen-4 (VLA-4; also called α(4)ß(1) integrin) is over-expressed on MM cells, and is one of the key mediators of myeloma cell adhesion to the bone marrow (BM) that promotes MM cell trafficking and drug resistance. Here we describe a proof-of-principle, novel molecular imaging strategy for MM tumors using a VLA-4 targeted PET radiopharmaceutical, (64)Cu-CB-TE1A1P-LLP2A. Cell uptake studies in a VLA-4-positive murine MM cell line, 5TGM1, demonstrated receptor specific uptake (P<0.0001, block vs. non-block). Tissue biodistribution at 2 h of (64)Cu-CB-TE1A1P-LLP2A in 5TGM1 tumor bearing syngeneic KaLwRij mice demonstrated high radiotracer uptake in the tumor (12±4.5%ID/g), and in the VLA-4 rich organs, spleen (8.8±1.0%ID/g) and marrow (11.6±2.0%ID/g). Small animal PET/CT imaging with (64)Cu-CB-TE1A1P-LLP2A demonstrated high uptake in the 5TGM1 tumors (SUV 6.6±1.1). There was a 3-fold reduction in the in vivo tumor uptake in the presence of blocking agent (2.3±0.4). Additionally, (64)Cu-CB-TE1A1P-LLP2A demonstrated high binding to the human MM cell line RPMI-8226 that was significantly reduced in the presence of the cold targeting agent. These results provide pre-clinical evidence that VLA-4-targeted imaging using (64)Cu-CB-TE1A1P-LLP2A is a novel approach to imaging MM tumors.