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The transcription factor NF-KB is sequestered in the cytoplasm by the inhibitor protein IKBc~. Extracellular inducers of NF-KB activate signal transduction pathways that result in the phosphorylation and subsequent degradation of IKBa. At present, the link between phosphorylation of IKB~x and its degradation is not understood. In this report we provide evidence that phosphorylation of serine residues 32 and 36 of IKBcx targets the protein to the ubiquitin-proteasome pathway. IKBa is ubiquitinated in vivo and in vitro following phosphorylation, and mutations that abolish phosphorylation and degradation of IKBa in vivo prevent ubiquitination in vitro. Ubiquitinated IgBa remains associated with NF-KB, and the bound IKBa is degraded by the 26S proteasome. Thus, ubiquitination provides a mechanistic link between phosphorylation and degradation of IKBr

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We have shown that thalidomide (Thal) and its immunomodulatory derivatives (IMiDs), proteasome inhibitor PS-341, and As 2 O 3 act directly on multiple myeloma (MM) cells and in the bone marrow (BM) milieu to overcome drug resistance. Although Thal/IMiDs, PS-341, and

Recent clinical trials using immunotherapy demonstrate its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies such as anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) or anti-Programmed cell death protein 1/anti-Programmed death-ligand 1 (anti-PD-1/anti-PD-L1)1 have demonstrated durable clinical responses in various cancers. Although these new immunotherapies have significant impact on cancer treatment, multiple mechanisms of immune resistance exist in tumors. Among the key mechanisms, myeloid cells play a major role in limiting effective tumor immunity. 2–4 Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. 5,6 These observations suggest a need for a precision medicine approach where the design of the immunotherapeutic combinations are tailored based on tumor immune landscape to overcome such resistance mechanisms. Herein we employ a preclinical model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumors. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3K-γ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3K–γ, with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumor immune microenvironment and promote cytotoxic T cell-mediated tumor regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3K-γ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumors.

Regulation of NF-B occurs through phosphorylation-dependent ubiquitination of IB␣, which is degradedNF-B is a transcription factor required for inducible expression of a number of proinflammatory mediators including cytokines, chemokines, and leukocyte adhesion molecules (6). In addition, NF-B regulates the expression of survival genes which prevent cell death in response to tumor necrosis factor alpha (TNF-␣) (7,37,59,62). NF-B is a member of the Rel family of proteins and is typically a heterodimer composed of p50 and p65 subunits. In quiescent cells, NF-B is retained in the cytosol bound to IB, a family of inhibitory proteins which mask the nuclear localization and DNA binding sequences on NF-B (5, 22). Stimulation of these cells with various cytokines, lipopolysaccharide, viruses, antigens, or oxidants triggers signaling events that ultimately lead to the phosphorylation and degradation of IB, allowing NF-B to translocate into the nucleus and activate target genes (3,21,38,54).Phosphorylation of Ser 32 and Ser 36 has been shown to target IB for ubiquitination and subsequent proteolysis by the ubiquitin-proteasome pathway (UPP) of protein degradation (2,8,45,49). The UPP is the principal pathway for intracellular protein turnover, including regulatory proteins (9). Protein substrates that enter the UPP are first marked by the covalent ligation of polyubiquitin chains mediated by a cascade of enzymes called E1 (ubiquitin activation enzyme), E2 (ubiquitinconjugating enzyme), and E3 (ubiquitin ligase) (9). In a reaction requiring ATP, ubiquitin is activated by E1 and charged onto an E2 through a thioester formed between the active-site cysteine residue in the E2 and the C-terminal glycine of ubiquitin. The E3 then directs the transfer of ubiquitin from the E2 onto lysine residues within specific substrate proteins, ultimately resulting in the formation of a ubiquitin-protein conjugate. Polyubiquitinated proteins are then recognized and degraded by the 26S proteasome complex to yield small peptides and monomeric ubiquitin.Recently, the receptor component of the IB E3 was identified as a member of the ␤TrCP (beta-transducin repeatcontaining protein) family of proteins called E3RS

Multiple cell adhesion proteins are up-regulated in vascular endothelial cells in response to TNF alpha and other inflammatory cytokines. This increase in cell adhesion gene expression is thought to require the transcription factor NF-kappa B. Here, we show that peptide aldehyde inhibitors of the proteasome, a multicatalytic protease recently shown to be required for the activation of NF-kappa B, block TNF alpha induction of the leukocyte adhesion molecules E-selectin, VCAM-1, and ICAM-1. Striking functional consequences of this inhibition were observed in analyses of leukocyte-endothelial interactions under defined flow conditions. Lymphocyte attachment to TNF alpha-treated endothelial monolayers was totally blocked, while neutrophil attachment was partially reduced but transmigration was essentially prevented.

SignificanceStandard of care for metastatic castration-resistant prostate cancer (mCRPC) mainly relies on suppression of androgen receptor (AR) signaling. This approach has no lasting benefit due to the emergence of resistance mechanisms, such as ligand-independent splicing variant AR-V7. A metabolic feature of mCRPC is the upregulation of de novo lipogenesis to provide substrates and fuel for metastatic spread. Whether increased levels of fats affect AR signaling to promote an aggressive disease remains to be determined. Using a selective and potent inhibitor of fatty acid synthase we demonstrate that suppression of this key enzyme inhibits AR, most importantly AR-V7, and reduces mCRPC growth. Our findings offer a therapeutic opportunity for mCRPC and a potential mechanism to overcome resistance to AR inhibitors.