Interactions of the BcI-2 protein with itself and other members of the Bcl-2 family, including Bcl-X-L, Bcl-X-S, Mci-i, and Bax, were explored with a yeast twohybrid system. Fusion proteins were created by linking BcI-2 family proteins to a LexA DNA-binding domain or a B42 trans-activation domain. Protein-protein interactions were examined by expression of these fusion proteins in Saccharomyces cerevisiae having a lacZ ((-galactosidase) gene under control of a LexA-dependent operator. This approach gave evidence for Bcl-2 protein homodimerization. Bcl-2 also interacted with Bcl-X-L and Mcl-i and with the dominant inhibitors Bax and Bcl-X-S. Bd-X-L displayed the same pattern of combinatorial interactions with Bd-2 family proteins as Bc1-2. Use of deletion mutants of Bc-2 suggested that BcI-2 homodimerization involves interactions between two distinct regions within the Bcl-2 protein, since a LexA protein containing Bcl-2 amino acids 83-218 mediated functional interactions with a B42 fusion protein contaiing Bcl-2 amino acids 1-81 but did not complement a B42 fusion protein containing BcI-2 amino acids 83-218. In contrast to LexA/Bcl-2 fusion proteins, expression of a LexA/Bax protein was lethal to yeast. This cytotoxicity could be abrogated by B42 fusion proteins containing BcI-2, Bcl-X-L, or Mci-i but not those containing Bcl-X-S (an alternatively spliced form of Bcl-X that lacks a well-conserved 63-amino acid region). The findings suggest a model whereby Bax and Bcl-X-S differentially regulate Bcd-2 function, and indicate that requirements for Bcl-2/Bax heterodimerization may be different from those for Bcl-2/Bcl-2 homodimerization.The bcl-2 gene becomes dysregulated in a wide variety of human cancers and contributes to neoplastic cell expansion by prolonging cell survival rather than by accelerating rates of cellular proliferation. Specifically, bcl-2 blocks programmed cell death, a physiological process that normally ensures a homeostatic balance between cell production and cell turnover in most tissues with self-renewal capacity and which often involves characteristic changes in cell morphology termed apoptosis. In fact, Bc1-2 can prevent or delay apoptosis induced by a wide variety of stimuli, including growth factor deprivation, alterations in Ca2+, free radicals, cytotoxic lymphokines, some types of viruses, radiation, and most chemotherapeutic drugs, suggesting that this oncoprotein controls a common final pathway involved in cell death regulation (reviewed in refs. 1 and 2).The mechanism by which Bcl-2 prevents cell death remains enigmatic, as the predicted amino acid sequence of the 26-kDa human Bcl-2 protein (239 aa) has no significant homology with other proteins whose biochemical activity is known. Recently, however, Bcl-2 has been shown to interact with a low molecular weight GTPase member of the Ras family, p23-R-Ras (3), and also can be coimmunoprecipitated with the serine/threonine-specific protein kinase Raf-1 (4). Thus, Bcl-2 may somehow regulate a signal transduction pathway involving...

Only a subset of cancer patients respond to T-cell checkpoint inhibitors, highlighting the need for alternative immunotherapeutics. We performed CRISPR-Cas9 screens in a leukemia cell line to identify perturbations that enhance natural killer effector functions. Our screens defined critical components of the tumor-immune synapse and highlighted the importance of cancer cell interferon-γ signaling in modulating NK activity. Surprisingly, disrupting the ubiquitin ligase substrate adaptor DCAF15 strongly sensitized cancer cells to NK-mediated clearance. DCAF15 disruption induced an inflamed state in leukemic cells, including increased expression of lymphocyte costimulatory molecules. Proteomic and biochemical analysis revealed that cohesin complex members were endogenous client substrates of DCAF15. Genetic disruption of DCAF15 was phenocopied by treatment with indisulam, an anticancer drug that functions through DCAF15 engagement. In AML patients, reduced DCAF15 expression was associated with improved survival. These findings suggest that DCAF15 inhibition may have useful immunomodulatory properties in the treatment of myeloid neoplasms.

Viral latency remains the most significant obstacle to HIV eradication. Clinical strategies aim to purge the latent CD4+ T cell reservoir by activating viral expression to induce death, but are undercut by the inability to target latently infected cells. Here we explored the acute signaling response of latent HIV-infected CD4+ T cells to identify dynamic phosphorylation signatures that could be targeted for therapy. Stimulation with CD3/CD28, PMA/ionomycin, or latency reversing agents prostratin and SAHA, yielded increased phosphorylation of IκBα, ERK, p38, and JNK in HIV-infected cells across two in vitro latency models. Both latent infection and viral protein expression contributed to changes in perturbation-induced signaling. Data-driven statistical models calculated from the phosphorylation signatures successfully classified infected and uninfected cells and further identified signals that were functionally important for regulating cell death. Specifically, the stress kinase pathways p38 and JNK were modified in latently infected cells, and activation of p38 and JNK signaling by anisomycin resulted in increased cell death independent of HIV reactivation. Our findings suggest that altered phosphorylation signatures in infected T cells provide a novel strategy to more selectively target the latent reservoir to enhance eradication efforts.

Human immunodeficiency virus 1 (HIV) latency remains a significant obstacle to curing infected patients. One promising therapeutic strategy is to purge the latent cellular reservoir by activating latent HIV with latency-reversing agents (LRAs). In some cases, co-drugging with multiple LRAs is necessary to activate latent infections, but few studies have established quantitative criteria for determining when co-drugging is required. Here we systematically quantified drug interactions between histone deacetylase inhibitors and transcriptional activators of HIV and found that the need for co-drugging is determined by the proximity of latent infections to the chromatin-regulated viral gene activation threshold at the viral promoter. Our results suggest two classes of latent viral integrations: those far from the activation threshold that benefit from co-drugging, and those close to the threshold that are efficiently activated by a single drug. Using a primary T cell model of latency, we further demonstrated that the requirement for co-drugging was donor dependent, suggesting that the host may set the level of repression of latent infections. Finally, we showed that single drug or co-drugging doses could be optimized, via repeat stimulations, to minimize unwanted side effects while maintaining robust viral activation. Our results motivate further study of patient-specific latency-reversing strategies.

Cellular senescence involves a stable cell cycle arrest coupled to a secretory program that, in some instances, stimulates the immune clearance of senescent cells. Using an immune competent liver cancer model in which senescence triggers CD8 T cell-mediated tumor rejection, we show that senescence also remodels the cell surface proteome to alter how tumor cells sense environmental factors, as exemplified by Type II interferon (IFN-y). Compared to proliferating cells, senescent cells upregulate the IFN-y receptor, become hypersensitized to microenvironmental IFN-y, and more robustly induce the antigen presenting machinery—-effects also recapitulated in human tumor cells undergoing therapy-induced senescence. Disruption of IFN-y sensing in senescent cells blunts their immune-mediated clearance without disabling the senescence state or its characteristic secretory program. Our results demonstrate that senescent cells have an enhanced ability to both send and receive environmental signals, and imply that each process is required for their effective immune surveillance.

Glucocorticoid administration is a well established cause of osteopenia. Mechanisms underlying the deleterious effect of glucocorticoids on bone may include direct inhibition of bone formation as well as indirect effects through changes in intestinal calcium absorption, renal calcium excretion, and/or levels of the calciotropic hormones. To further examine the potential role of the calciotropic hormones we measured serum levels of PTH and 1,25 dihydroxyvitamin D [1,25(OH)2D], as well as serum and urine levels of calcium and vertebral bone density in patients with chronic obstructive pulmonary disease being managed with or without prednisone. Patients treated with prednisone had lower spinal bone density (53 vs. 106 mg/cm3) and higher serum calcium (2.40 vs. 2.33 mmol/l), urine calcium (6.9 vs. 2.7 mmol/24h), and 1,25(OH)2D levels (147 vs. 95 pmol/L). Compared to the patients not treated with glucocorticoids. PTH levels also tended to be higher (33 vs. 26 microliters-eq/ml), but the difference was not significant. Serum and urine calcium levels correlated positively with 1,25(OH)2D levels, but none of these measurements correlated with PTH levels. Our results suggest that prednisone treatment alters the regulation of 1,25(OH)2D production, and this may contribute to the loss of bone mineral induced by prednisone.

12Only a subset of cancer patients respond to T-cell checkpoint inhibitors, highlighting the need for 13 alternative immunotherapeutics. We performed CRISPR-Cas9 screens in a leukemia cell line to 14 identify perturbations that enhance natural killer effector functions. Our screens defined critical 15 components of the tumor-immune synapse and highlighted the importance of cancer cell 16 interferon-g signaling in modulating NK activity. Surprisingly, disrupting the ubiquitin ligase 17 substrate adaptor DCAF15 strongly sensitized cancer cells to NK-mediated clearance. DCAF15 18 disruption induced an inflamed state in leukemic cells, including increased expression of 19 lymphocyte costimulatory molecules. Proteomic and biochemical analysis revealed that cohesin 20 complex members were endogenous client substrates of DCAF15. Genetic disruption of DCAF15 21 was phenocopied by treatment with indisulam, an anticancer drug that functions through DCAF15 22 engagement. In AML patients, reduced DCAF15 expression was associated with improved 23 survival. These findings suggest that DCAF15 inhibition may have useful immunomodulatory 24 properties in the treatment of myeloid neoplasms. 25 26 T and NK cells share effector functions, releasing cytokines and exocytosing lytic granules upon 58 activation to kill target cells. However, NK activation status is controlled by the integrated signals 59 from germline-encoded NK-activating and -inhibiting receptors (aNKRs/iNKRs). Generally, 60 iNKR ligands are expressed by normal and healthy cells, whereas aNKR ligands are upregulated 61 after DNA damage or viral insult 19,20 . MHC-I molecules provide a potent inhibitory signal sensed 62 by NK cells, enabling the innate immune system to respond productively to MHC-deficient cells. 63As a result, there is considerable interest in amplifying NK responses to cancers, as well as 64 developing NK-based cell therapies [18][19][20] . 65 66 Here, we performed genetic screens in an MHC-deficient leukemic cell line to systematically 67 identify modulators of NK-mediated anti-cancer immunity. These screens, unexpectedly, revealed 68 the potential therapeutic utility of targeting the CRL4 substrate adaptor DCAF15 in myeloid 69 malignancies. Disruption of DCAF15 strongly sensitized cancer cells to NK-mediated killing, 70 resulting from increased cancer cell expression of lymphocyte costimulatory molecules. Proteomic 71 experiments revealed that DCAF15 interacted with and promoted the ubiqitination of the cohesin 72 complex members. Treatment with indisulam, an anticancer drug that modulates DCAF15 73 function, reduced interaction with cohesin members and mimicked DCAF15 loss-of-function 74 immunophenotypes. 75 76 Results 77 78 A genome-scale CRISPR screen identifies modulators of NK effector functions 79 80We performed genome-scale CRISPR screens in K562 cells to identify perturbations that modulate 81 NK-92-mediated killing ( Figure 1A). K562 human chronic myelogenous leukemia cells are a NK-82 sensitive cancer cell line that weakly expresses MHC-...

Conventional emulsion‐based sunscreen formulations are limited by postapplication epicutaneous penetration that increases the risk of allergic dermatitis, cellular damage, and filter photodegradation upon ultraviolet radiation (UVR) exposure. Encapsulation of the UVB filter padimate O within bioadhesive biodegradable nanoparticles (BNPs) composed of poly(d,l‐lactic acid)‐hyperbranched polyglycerol was previously shown to enhance UVR protection while preventing skin absorption. Herein, we assess the capacity of BNP co‐incorporation of avobenzone and octocrylene to provide broad‐spectrum UVR protection. The ratio of UV filters within nanoparticles (NPs) was optimized for filter–filter stabilization upon UV irradiation and maximum drug loading. In vitro water‐resistance test showed significant particle retention at 85% over 3 hr. In a pilot clinical study, protection against UVR‐induced erythema of BNPs was found to be comparable to the FDA standard P2. Thus, sunscreen formulations utilizing BNP incorporation of a combination of organic filters may offer key safety and performance advantages.