Pre-clinical validation of B cell maturation antigen (BCMA) as a target for T cell immunotherapy of multiple myeloma.

Multiple myeloma has a continued need for more effective and durable therapies. B cell maturation antigen (BCMA), a plasma cell surface antigen and member of the tumor necrosis factor (TNF) receptor superfamily, is an attractive target for immunotherapy of multiple myeloma due to its high prevalence on malignant plasma cells. The current work details the pre-clinical evaluation of BCMA expression and development of a chimeric antigen receptor (CAR) targeting this antigen using a fully human single chain variable fragment (scFv). We demonstrate that BCMA is prevalently, but variably expressed by all MM with expression on 25-100% of malignant plasma cells. Extensive Immunohistochemical analysis of normal tissue expression using commercially available polyclonal antibodies demonstrated expression within B-lineage cells across a number of tissues as expected. Based upon the highly restricted expression of BCMA within normal tissues, we generated a set of novel, fully human scFv binding domains to BCMA by screening a naïve B-cell derived phage display library. Using a series of in vitro and pre-clinical in vivo studies, we identified a scFv with high specificity for BCMA and robust anti-myeloma activity when used as the binding domain of a second-generation CAR bearing a CD137 costimulatory domain. This BCMA-specific CAR is currently being evaluated in a Phase 1b clinical study in relapsed and refractory MM patients (NCT02546167).

SKAR links pre-mRNA splicing to mTOR/S6K1-mediated enhanced translation efficiency of spliced mRNAs.

Different protein complexes form on newly spliced mRNA to ensure the accuracy and efficiency of eukaryotic gene expression. For example, the exon junction complex (EJC) plays an important role in mRNA surveillance. The EJC also influences the first, or pioneer round of protein synthesis through a mechanism that is poorly understood. We show that the nutrient-, stress-, and energy-sensing checkpoint kinase, mTOR, contributes to the observed enhanced translation efficiency of spliced over nonspliced mRNAs. We demonstrate that, when activated, S6K1 is recruited to the newly synthesized mRNA by SKAR, which is deposited at the EJC during splicing, and that SKAR and S6K1 increase the translation efficiency of spliced mRNA. Thus, SKAR-mediated recruitment of activated S6K1 to newly processed mRNPs serves as a conduit between mTOR checkpoint signaling and the pioneer round of translation when cells exist in conditions supportive of protein synthesis.

SKAR is a specific target of S6 kinase 1 in cell growth control.

BACKGROUND: The mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K) signaling pathways promote cell growth and cell cycle progression in response to nutritional, energy, and mitogenic cues. In mammalian cells, the ribosomal protein S6 kinases, S6K1 and S6K2, lie downstream of mTOR and PI3K, suggesting that translational control through the phosphorylation of S6 regulates cell growth. Interestingly, genetic experiments predict that a substrate that is specific to S6K1 but not S6K2 regulates cell growth. RESULTS: Here we identify SKAR as a novel and specific binding partner and substrate of S6K1 but not S6K2. We find that serines 383 and 385 of human SKAR are insulin-stimulated and rapamycin-sensitive S6K1 phosphorylation sites. Quantitative mass spectrometry reveals that serine 383/385 phosphorylation is sensitive to RNA interference (RNAi)-mediated S6K1 reduction, but not S6K2 reduction. Furthermore, RNAi-mediated reduction of SKAR decreases cell size. SKAR is nuclear protein with homology to the Aly/REF family of RNA binding proteins, which has been proposed to couple transcription with pre-mRNA splicing and mRNA export. CONCLUSIONS: We have identified a novel and specific target of S6K1, SKAR, which regulates cell growth. The homology of SKAR to the Aly/REF family links S6K1 with mRNA biogenesis in the control of cell growth.

PI3-kinase and TOR: PIKTORing cell growth.

Regulation of growth and proliferation in higher eukaryotic cells results from an integration of nutritional, energy, and mitogenic signals. Biochemical processes underlying cell growth and proliferation are governed by the phosphatidylinositol 3-kinase (PI3K) and target of rapamycin (TOR) signaling pathways. The importance of the interplay between these two pathways is underscored by the discovery that the TOR inhibitor rapamycin is effective against tumors caused by misregulation of the PI3K pathway. We review here recent data concerning the convergence of the PI3K and TOR pathways, the role of these pathways in cell growth and proliferation, and the regulation of growth by downstream TOR targets.

mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E.

The mammalian target of rapamycin (mTOR) integrates nutrient and mitogen signals to regulate cell growth (increased cell mass and cell size) and cell division. The immunosuppressive drug rapamycin inhibits cell cycle progression via inhibition of mTOR; however, the signaling pathways by which mTOR regulates cell cycle progression have remained poorly defined. Here we demonstrate that restoration of mTOR signaling (by using a rapamycin-resistant mutant of mTOR) rescues rapamycin-inhibited G(1)-phase progression, and restoration of signaling along the mTOR-dependent S6K1 or 4E-BP1/eukaryotic translation initiation factor 4E (eIF4E) pathways provides partial rescue. Furthermore, interfering RNA-mediated reduction of S6K1 expression or overexpression of mTOR-insensitive 4E-BP1 isoforms that block eIF4E activity inhibit G(1)-phase progression individually and additively. Thus, the activities of both the S6K1 and 4E-BP1/eIF4E pathways are required for and independently mediate mTOR-dependent G(1)-phase progression. In addition, overexpression of constitutively active mutants of S6K1 or wild-type eIF4E accelerates serum-stimulated G(1)-phase progression, and stable expression of wild-type S6K1 confers a proliferative advantage in low-serum-containing media, suggesting that the activity of each of these pathways is limiting for cell proliferation. These data demonstrate that, as for the regulation of cell growth and cell size, the S6K1 and 4E-BP1/eIF4E pathways each represent critical mediators of mTOR-dependent cell cycle control.