Jayakumar Nair

Sustained antibody responses depend on CD28 function in bone marrow-resident plasma cells.

CD28 signaling is essential for maintenance of long-term antigen-specific antibody production and for persistence of plasma cells in the bone marrow of mice.

CD28 Expressed on Malignant Plasma Cells Induces a Prosurvival and Immunosuppressive Microenvironment

Interactions between the malignant plasma cells of multiple myeloma and stromal cells within the bone marrow microenvironment are essential for myeloma cell survival, mirroring the same dependence of normal bone marrow-resident long-lived plasma cells on specific marrow niches. These interactions directly transduce prosurvival signals to the myeloma cells and also induce niche production of supportive soluble factors. However, despite their central importance, the specific molecular and cellular components involved remain poorly characterized. We now report that the prototypic T cell costimulatory receptor CD28 is overexpressed on myeloma cells during disease progression and in the poor-prognosis subgroups and plays a previously unrecognized role as a two-way molecular bridge to support myeloid stromal cells in the microenvironment. Engagement by CD28 to its ligand CD80/CD86 on stromal dendritic cell directly transduces a prosurvival signal to myeloma cell, protecting it against chemotherapy and growth factor withdrawal-induced death. Simultaneously, CD28-mediated ligation of CD80/CD86 induces the stromal dendritic cell to produce the prosurvival cytokine IL-6 (involving novel cross-talk with the Notch pathway) and the immunosuppressive enzyme IDO. These findings identify CD28 and CD80/CD86 as important molecular components of the interaction between myeloma cells and the bone marrow microenvironment, point to similar interaction for normal plasma cells, and suggest novel therapeutic strategies to target malignant and pathogenic (e.g., in allergy and autoimmunity) plasma cells.

Novel regulation of CD80/CD86-induced phosphatidylinositol 3-kinase signaling by NOTCH1 protein in interleukin-6 and indoleamine 2,3-dioxygenase production by dendritic cells.

Background: Engagement of CD80/CD86 on dendritic cells by CD28 on T cells induces dendritic cell production of IL-6 and IDO. Results: The NOTCH pathway modulates activation of the PI3K pathway downstream of CD80/CD86 ligation and regulates IL-6 and IDO production. Conclusion: Cross-talk between NOTCH and PI3K pathways modulates dendritic cell production of IL-6 and IDO. Significance: Elucidating the molecular mechanism of NOTCH-PI3K cross-talk will have broad implications in human disease. Dendritic cells (DC) play a critical role in modulating antigen-specific immune responses elicited by T cells via engagement of the prototypic T cell costimulatory receptor CD28 by the cognate ligands CD80/CD86, expressed on DC. Although CD28 signaling in T cell activation has been well characterized, it has only recently been shown that CD80/CD86, which have no demonstrated binding domains for signaling proteins in their cytoplasmic tails, nonetheless also transduce signals to the DC. Functionally, CD80/CD86 engagement results in DC production of the pro-inflammatory cytokine IL-6, which is necessary for full T cell activation. However, ligation of CD80/CD86 by CTLA4 also induces DC production of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO), which depletes local pools of the essential amino acid tryptophan, resulting in blockade of T cell activation. Despite the significant role of CD80/CD86 in immunological processes and the seemingly opposing roles they play by producing IL-6 and IDO upon their activation, how CD80/CD86 signal remains poorly understood. We have now found that cross-linking CD80/CD86 in human DC activates the PI3K/AKT pathway. This results in phosphorylation/inactivation of its downstream target, FOXO3A, and alleviates FOXO3A-mediated suppression of IL-6 expression. A second event downstream of AKT phosphorylation is activation of the canonical NF-κB pathway, which induces IL-6 expression. In addition to these downstream pathways, we unexpectedly found that CD80/CD86-induced PI3K signaling is regulated by previously unrecognized cross-talk with NOTCH1 signaling. This cross-talk is facilitated by NOTCH-mediated up-regulation of the expression of prolyl isomerase PIN1, which in turn increases enzyme activity of casein kinase II. Subsequently, phosphatase and tensin homolog (which suppresses PI3K activity) is inactivated via phosphorylation by casein kinase II. This results in full activation of PI3K signaling upon cross-linking CD80/CD86. Similar to IL-6, we have found that CD80/CD86-induced IDO production by DC at late time points is also dependent upon the PI3K → AKT → NF-κB pathway and requires cross-talk with NOTCH signaling. These data further suggest that the same signaling pathways downstream of DC CD80/CD86 cross-linking induce early IL-6 production to enhance T cell activation, followed by later IDO production to self-limit this activation. In addition to characterizing the pathways downstream of CD80/CD86 in IL-6 and IDO production, identification of a novel cross-talk between NOTCH1 and PI3K signaling may provide new insights in other biological processes where PI3K signaling plays a major role.

KLF9 is a novel transcriptional regulator of bortezomib- and LBH589-induced apoptosis in multiple myeloma cells

Bortezomib, a therapeutic agent for multiple myeloma (MM) and mantle cell lymphoma, suppresses proteosomal degradation leading to substantial changes in cellular transcriptional programs and ultimately resulting in apoptosis. Transcriptional regulators required for bortezomib-induced apoptosis in MM cells are largely unknown. Using gene expression profiling, we identified 36 transcription factors that displayed altered expression in MM cells treated with bortezomib. Analysis of a publically available database identified Kruppel-like family factor 9 (KLF9) as the only transcription factor with significantly higher basal expression in MM cells from patients who responded to bortezomib compared with nonresponders. We demonstrated that KLF9 in cultured MM cells was up-regulated by bortezomib; however, it was not through the induction of endoplasmic reticulum stress. Instead, KLF9 levels correlated with bortezomib-dependent inhibition of histone deacetylases (HDAC) and were increased by the HDAC inhibitor LBH589 (panobinostat). Furthermore, bortezomib induced binding of endogenous KLF9 to the promoter of the proapoptotic gene NOXA. Importantly, KLF9 knockdown impaired NOXA up-regulation and apoptosis caused by bortezomib, LBH589, or a combination of theses drugs, whereas KLF9 overexpression induced apoptosis that was partially NOXA-dependent. Our data identify KLF9 as a novel and potentially clinically relevant transcriptional regulator of drug-induced apoptosis in MM cells.

CD28-mediated pro-survival signaling induces chemotherapeutic resistance in multiple myeloma

Chemotherapeutic resistance remains a significant hurdle in the treatment of multiple myeloma (MM) and is significantly mediated by interactions between MM cells and stromal cells of the bone marrow microenvironment. Despite the importance of these interactions, the specific molecules and downstream signaling components involved remain incompletely understood. We have previously shown that the prototypic T-cell costimulatory receptor CD28, which is also expressed on MM cells, is a key mediator of MM survival and apoptotic resistance. Crosslinking CD28 by agonistic antibodies or myeloid dendritic cells (DC; these express the CD28 ligands CD80/CD86) prevents apoptosis caused by chemotherapy or serum withdrawal. We now report that CD28 pro-survival signaling is dependent upon downstream activation of phosphatidyl-inositol 3-kinase/Akt, inactivation of the transcription factor FoxO3a, and decreased expression of the pro-apoptotic molecule Bim. Conversely, blocking the CD28-CD80/CD86 interaction between MM cells and DC in vitro abrogates the DCs ability to protect MM cells against chemotherapy-induced death. Consistent with these observations, in vivo blockade of CD28-CD80/CD86 in the Vk*MYC murine myeloma model sensitizes MM cells to chemotherapy and significantly reduces tumor burden. Taken together, our findings suggest that CD28 is an important mediator of MM survival during stress and can be targeted to overcome chemotherapy resistance.

CD28 Promotes Plasma Cell Survival, Sustained Antibody Responses, and BLIMP-1 Upregulation through Its Distal PYAP Proline Motif

In health, long-lived plasma cells (LLPC) are essential for durable protective humoral immunity, and, conversely, in disease are a major source of pathogenic Abs in autoimmunity, graft rejection, and allergy. However, the molecular basis for their longevity is largely unknown. We have recently found that CD28 signaling in plasma cells (PC) is essential for sustaining Ab titers, by supporting the survival of LLPC, but not short-lived PC (SLPC). We now find that, unlike SLPC, CD28 activation in LLPC induces prosurvival downstream Vav signaling. Knockin mice with CD28 cytoplasmic tail mutations that abrogate Vav signaling (CD28-AYAA) had significantly fewer LLPC but unaffected SLPC numbers, whereas mice with mutations that abrogate PI3K signaling (CD28-Y170F) were indistinguishable from wild-type controls. This was consistent with the loss of CD28’s prosurvival effect in LLPC from CD28-AYAA, but not CD28-Y170F, mice. Furthermore, the CD28 Vav motif in the B lineage was essential for the long-term maintenance of Ag-specific LLPC populations and Ab titers in vivo. Signaling downstream of the CD28 Vav motif induced previously undescribed transcriptional regulation of B lymphocyte–induced maturation protein-1, a key mediator of PC differentiation and maintenance. These findings suggest CD28 signaling in LLPC modulates the central B lymphocyte–induced maturation protein-1 transcriptional nexus involved in long-term survival and function.

Comparative genomic analysis reveals a novel mitochondrial isoform of human rTS protein and unusual phylogenetic distribution of the rTS gene

BackgroundThe rTS gene (ENOSF1), first identified in Homo sapiens as a gene complementary to the thymidylate synthase (TYMS) mRNA, is known to encode two protein isoforms, rTSα and rTSβ. The rTSβ isoform appears to be an enzyme responsible for the synthesis of signaling molecules involved in the down-regulation of thymidylate synthase, but the exact cellular functions of rTS genes are largely unknown.ResultsThrough comparative genomic sequence analysis, we predicted the existence of a novel protein isoform, rTS, which has a 27 residue longer N-terminus by virtue of utilizing an alternative start codon located upstream of the start codon in rTSβ. We observed that a similar extended N-terminus could be predicted in all rTS genes for which genomic sequences are available and the extended regions are conserved from bacteria to human. Therefore, we reasoned that the protein with the extended N-terminus might represent an ancestral form of the rTS protein. Sequence analysis strongly predicts a mitochondrial signal sequence in the extended N-terminal of human rTSγ, which is absent in rTSβ. We confirmed the existence of rTS in human mitochondria experimentally by demonstrating the presence of both rTSγ and rTSβ proteins in mitochondria isolated by subcellular fractionation. In addition, our comprehensive analysis of rTS orthologous sequences reveals an unusual phylogenetic distribution of this gene, which suggests the occurrence of one or more horizontal gene transfer events.ConclusionThe presence of two rTS isoforms in mitochondria suggests that the rTS signaling pathway may be active within mitochondria. Our report also presents an example of identifying novel protein isoforms and for improving gene annotation through comparative genomic analysis.

CD28 - OLD DOG, NEW TRICKS: CD28 IN PLASMA CELL/MULTIPLE MYELOMA BIOLOGY

The concept of a costimulatory signal requirement for immune cell activation has been attributed to Bretscher and Cohn, 7 who first proposed that B cell activation required two signals. This model was subsequently modified by Lafferty and Cunningham for T cell activation and allograft rejection. 7 , 34 For naive T cell activation, signal one is delivered by the T cell receptor (TCR) binding to cognate antigen presented by major histocompatibility complex (MHC) molecules on professional antigen presentation cells (APCs). The second signal is delivered by costimulatory receptor binding to its ligand(s) on the APC. While the costimulatory signal alone characteristically has no effect on T cells, in combination with a signal 1 it has been clearly shown to enhance cytokine secretion, proliferation, metabolic fitness, and survival during T cell activation. The prototypic costimulatory receptor is CD28, and its effect on T cell activation has been extensively characterized. However, CD28 was also initially described to be expressed on normal plasma cells but not B cells, 29 and subsequently on the transformed counterparts of these cells (namely, multiple myeloma) – where it has been shown to be significantly associated with disease progression and poor prognosis. 14 , 52 , 56 However, plasma cells (both normal and transformed) no longer express an antigen-specific receptor to deliver a signal 1, and the biological role of CD28 in the B cell lineage has been unclear. Yet CD28 expression is highly regulated (suppressed) in B cells by Pax5, a key transcriptional regulator of mature B cell → plasma cell differentiation, 13 supporting a role for CD28 in plasma cell biology. Because multiple myeloma (MM) cells (like normal PC) are critically dependent on (largely undefined) cell–cell interactions with the bone marrow microenvironment, these interactions are central in developing new therapeutic targets for this incurable disease. This review will examine the role of CD28 expressed on PC/MM cells as a key component of this interaction.

Putting the brakes on angiogenesis through a novel VEGF–KLH (kinoid) vaccine

Evaluation of: Rad FH, Buanec HL, Paturance S et al. VEGF–kinoid vaccine, a therapeutic approach against tumor angiogenesis and metastases. Proc. Natl Acad. Sci. USA 104(8), 2837–2842 (2007). Angiogenesis, the growth of new blood vessels, is essential for tumor growth and metastasis. Of the several known angiogenic factors, VEGF is an important mediator of tumor-induced angiogenesis and represents a potential target for innovative anticancer therapy. Recently, humanized monoclonal anti-VEGF antibody (bevacizumab) has been approved by the US FDA for combinatorial therapies with cytotoxic drugs in metastatic colorectal cancer. However, adverse side effects and enormous costs are associated with the use and delivery of bevacizumab. In the study under evaluation, Rad et al. demonstrated an alternative approach by using active immunization in mice with a novel VEGF–kinoid vaccine. The authors observed that the antitumor effects elicited by their vaccine were as effective as bevacizumab in xenografted-tumor mouse models.

Abstract 4854: Evaluating HDAC6 as a causal factor in metastatic breast cancer to develop immunotherapy

Histone deacetylases (HDACs), originally described as histone modifiers, have recently been demonstrated to modify a variety of other proteins involved in diverse cellular processes unrelated to the chromatin environment, including the modulation of proteins related to cell cycle/apoptosis and immune regulation. In contrast to the well-documented effects of HDAC inhibitors (HDACi) in the control of cell cycle and apoptosis, their role in immune-biology is incompletely understood. We have found that the pharmacological or genetic abrogation of a single HDAC, i.e. HDAC6, inactivates the STAT3 pathway and modulates the expression of immuno-regulatory proteins, including the down-regulation of PD-L1, PD-L2 and B7-H4, important negative regulators of immune function, often over-expressed in cancer cells; including breast cancer. HDAC6 has been also involved in a number of structural functions related to cellular motility, shape and intracellular transport through the regulation of the acetylation of numerous targets, including tubulin and cortactin. This function is strongly suggestive of HDAC6 being a key player in metastatic cancer progression. In our initial studies we observed that the selective HDAC6 inhibitor Nexturastat A is capable of reducing the tumor growth in a highly aggressive murine mammary carcinoma that mimics human triple negative breast cancer (TNBC), under both orthotopic and subcutaneous conditions of implantation. Additionally, we observed that the size and number of secondary tumor nodules in lungs were significantly diminished after the HDAC6i treatment. In order to boost the anti-tumor T-cell response, we also tested check-point inhibitors against the tumor (such as anti PD-1 and CTLA4 antibodies). While each of the standalone treatments showed a certain degree of success in reducing tumor growth and enhancing intra-tumoral IFNγ, we demonstrated that HDAC6i improves anti-tumor immune responses when combined with immune check-point blockade. Citation Format: Debarati Banik, Melissa Hadley, Jennifer Kim, Tessa Knox, Jayakumar Nair, Alan Kozikowski, Sida Shen, Charu Vyas, Ashleyn Donohue, Eduardo Sotomayor, Alejandro Villagra. Evaluating HDAC6 as a causal factor in metastatic breast cancer to develop immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4854. doi:10.1158/1538-7445.AM2017-4854