Intramural Research Program of the National Institutes of Health and National Cancer Institute.

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.

Purpose: Preclinical studies suggest PARP inhibition (PARPi) induces immunostimulatory micromilieu in ovarian cancer thus complementing activity of immune checkpoint blockade. We conducted a phase II trial of PARPi olaparib and anti-PD-L1 durvalumab and collected paired fresh core biopsies and blood samples to test this hypothesis.Patients and Methods: In a single-center, proof-of-concept phase II study, we enrolled women aged ≥18 with recurrent ovarian cancer. All patients were immune checkpoint inhibitor-na€ ve and had measurable disease per RECISTv1.1, ECOG performance status 0-2, and adequate organ and marrow function. Patients received olaparib 300 mg twice daily and durvalumab 1,500 mg intravenously every 4 weeks until disease progression, unacceptable toxicity, or withdrawal of consent. Primary endpoint was overall response rate (ORR). Secondary objectives were safety and progression-free survival (PFS). Translational objectives included biomarker evaluation for relationships with clinical response and immunomodulatory effects by treatment.Results: Thirty-five patients with ovarian cancer [median, four prior therapies (IQR, 2-5.5), predominantly platinum-resistant (86%), BRCA wild-type (77%)] received at least one full cycle of treatment. ORR was 14% [5/35; 95% confidence interval (CI), 4.8%-30.3%]. Disease control rate (PRþSD) was 71% (25/35; 95% CI, 53.7%-85.4%). Treatment enhanced IFNg and CXCL9/CXCL10 expression, systemic IFNg/TNFa production, and tumorinfiltrating lymphocytes, indicating an immunostimulatory environment. Increased IFNg production was associated with improved PFS [HR, 0.37 (95% CI, 0.16-0.87), P ¼ 0.023], while elevated VEGFR3 levels were associated with worse PFS (HR, 3.22 (95% CI, 1.23-8.40), P ¼ 0.017].Conclusions: The PARPi and anti-PD-L1 combination showed modest clinical activity in recurrent ovarian cancer. Our correlative study results suggest immunomodulatory effects by olaparib/ durvalumab in patients and indicate that VEGF/VEGFR pathway blockade would be necessary for improved efficacy of the combination.

Abstract. SEC2 function is required at the post-Golgi apparatus stage of the yeast secretory pathway. The SEC2 sequence encodes a protein product of 759 amino acids containing an amino terminal region that is predicted to be in an c~-helical, coiled-coil conformation. Two temperature-sensitive alleles, sec2-41 and sec2-59, encode proteins truncated by opal stop codons and are suppressible by an opal tRNA suppressor. Deletion analysis indicates that removal of the carboxy terminal 251 amino acids has no apparent phenotype, while truncation of 368 amino acids causes temperature sensitivity. The amino terminal half of the protein, containing the putative coiled-coil domain, is essential at all temperatures. Sec2 protein is found predominantly in the soluble fraction and displays a native molecular mass of >500 kD. All phenotypes of the temperature-sensitive sec2 alleles are partially suppressed by duplication of the SEC4 gene, but the lethality of a sec2 disruption is not suppressed. The sec2-41 mutation exhibits synthetic lethality with the same subset of the late acting sec mutants as does sec4-8 and secl5-1. The Sec2 protein may function in conjunction with the Sec4 and Secl5 proteins to control vesicular traffic.

Interactions between the malignant plasma cells of multiple myeloma (MM) and stromal cells within the bone marrow (BM) microenvironment are essential for myeloma cell survival, mirroring the same dependence of normal BM-resident long-lived plasma cells on specific marrow niches. These interactions directly transduce pro-survival 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 (DC) directly transduces a pro-survival signal to myeloma cell, protecting it against chemotherapy and growth factor withdrawal-induced death. Simultaneously, CD28-mediated ligation of CD80/CD86 induces the stromal DC to produce the pro-survival cytokine IL-6 (involving novel crosstalk with the Notch pathway) and the immunosuppressive enzyme indoleamine 2, 3 dioxygenase (IDO). These findings identify CD28 and CD80/CD86 as important molecular components of the interaction between myeloma cells and the bone marrow microenvironment, and point to similar interaction for normal plasma cells as well as suggesting novel therapeutic strategies to target malignant and pathogenic (e.g. in allergy and autoimmunity) plasma cells.

• CD28 delivers a pro-survival signal to MM cells via regulation of PI3K/Akt, FoxO3a, and Bim.• Blockade of CD28:CD80/ CD86 in vivo resensitizes MM cells to chemotherapy and significantly reduces tumor burden.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 DC's ability to protect MM cells against chemotherapyinduced 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. (Blood. 2014;123(24): 3770-3779) IntroductionMultiple myeloma (MM), the bone marrow (BM)-resident plasma cell (PC) neoplasm, is the second most common hematologic malignancy after non-Hodgkin lymphoma.1 Although new therapies have improved survival, MM remains almost uniformly fatal and only curable in a small fraction of patients. 2,3 Initially, patients are responsive to therapy and experience remission; however, relapses result in MM cells that are progressively resistant to therapy. 3,4 Thus, understanding and overcoming resistance mechanisms may lead to development of new therapeutic approaches.Chemotherapies such as the DNA alkylator melphalan and the proteasome inhibitor bortezomib were developed because of their direct apoptotic effects on MM cells. 5,6 However, these agents, thalidomide, and thalidomide derivatives also target the BM microenvironment, pointing to the key role that stroma plays in myeloma survival.6-8 Moreover, primary MM culture in vitro requires stroma, indicating that the BM niche provides essential pro-survival signals.9-11 Thus, identifying key interactions between MM and the microenvironment is essential for understanding and overcoming therapeutic resistance mechanisms.Broadly, MM-stromal interactions fall into 2 categories. The first consists of soluble pro-survival factors induced from stromal niche cells upon MM interaction, and include int...

PARP inhibitors (PARPi) have been effective in high-grade serous ovarian cancer (HGSOC), although clinical activity is limited against BRCA wild type HGSOC. The nearly universal loss of normal p53 regulation in HGSOCs causes dysfunction in the G1/S checkpoint, making tumor cells reliant on Chk1-mediated G2/M cell cycle arrest for DNA repair. Therefore, Chk1 is a reasonable target for a combination strategy with PARPi in treating BRCA wild type HGSOC. Here we investigated the combination of prexasertib mesylate monohydrate (LY2606368), a Chk1 and Chk2 inhibitor, and a PARP inhibitor, olaparib, in HGSOC cell lines (OVCAR3, OV90, PEO1 and PEO4) using clinically attainable concentrations. Our findings showed combination treatment synergistically decreased cell viability in all cell lines and induced greater DNA damage and apoptosis than the control and/or monotherapies (p<0.05). Treatment with olaparib in BRCA wild type HGSOC cells caused formation of Rad51 foci, whereas the combination treatment with prexasertib inhibited transnuclear localization of Rad51, a key protein in homologous recombination repair. Overall, our data provide evidence that prexasertib and olaparib combination resulted in synergistic cytotoxic effects against BRCA wild type HGSOC cells through reduced Rad51 foci formation and greater induction of apoptosis. This may be a novel therapeutic strategy for HGSOC.

182 Protective immunity against infection requires sustained antibody production by long-lived plasma cells (LLPC) that survive for years/decades within specialized niches. What regulates/supports this survival remains largely unknown. However, it has been shown that normal and transformed (human multiple myeloma) LLPC are critically dependent on the bone marrow microenvironment, including cell-to-cell interactions. This lead us to hypothesize that modulating these interactions could either enhance antibody production for vaccine development or, conversely, compromise the survival of transformed/normal LLPC in the bone marrow microenvironment. We have shown that the T cell costimulatory receptor CD28 expressed on both normal and transformed LLPC, plays an essential role in survival. While LLPC and short-lived plasma cells (SLPC) both express CD28, its activation in vitro only significantly increases survival and IgG production in LLPC. Consistent with these findings, we show in vivo, vaccinated bone marrow CD28−/−:μMT chimeras had significantly reduced long-term antibody titers and decreased LLPC (but not SLPC) t1/2 from 426 to 63 days. These findings demonstrate the existence of a distinct bone marrow (BM) LLPC subset necessary to sustain antibody titers, and establish a central role for CD28 function in the maintenance of plasma cells and humoral immunity. While CD28 signaling has been shown to play an important role in maintaining long-term humoral immune responses, the mechanism by which CD28 signaling affects PC function has not yet been determined. To further elucidate CD28 signaling in BM PC, we utilized CD28 conditional knock-in mice. In these mice, the CD28 cytoplasmic tail is mutated at either the YMNM or proline-rich motifs, resulting in an inhibition of PI3K or vav signaling, respectively. We found that CD28-vav signaling deficient BM PC were selectively depleted in vivo and could not be rescued by CD28 activation in in vitro serum starvation conditions. Furthermore, anti-CD28 mAb drove a 1.5 fold increase in Blimp-1 expression in BM PC, compared to control. This increase was regulated through the CD28-vav signaling pathway, as CD28 activation in CD28-vav signaling deficient BM PC did not increase Blimp-1 expression. To further determine if CD28 is acting directly on the Blimp-1 promoter, we examined in silico for a CD28RE composite element, previously reported to transcriptionally regulate IL-2 production in T cells and IL-8 production in myeloma cells. To our surprise, we found a CD28RE “like” site 4712bp upstream of the Blimp-1 start site. To confirm CD28 transcriptionally regulates Blimp-1 promoter activity, we transfected the CD28+ plasmacytoma cell line J558 with full-length or truncated Blimp-1 promoter constructs (i.e. 7000bp, 4500bp, 1500bp). We found CD28 activation enhances Blimp-1 activity in J558 cells transfected with full-length-Blimp-1, and this activity was lost when the promoter was truncated. Using site-directed mutagenesis, we confirmed the CD28RE is required for induction of Blimp-1 in PC. Furthermore, we show CD28 activation of Blimp-1 increases the BCMA receptor in BM PC. Taken together, our data suggests the CD28-vav signaling pathway in PC induces a CD28RE composite element, which is necessary for the induction of the key PC transcriptional regulator Blimp-1, required to maintain LLPC and humoral immunity. Disclosures: No relevant conflicts of interest to declare.