Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response. Inositolrequiring enzyme 1␣ (IRE1␣) is activated to splice X-box binding protein 1 (XBP1) mRNA, thereby increasing XBP1s protein, which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study, we examined whether IRE1␣- IntroductionTreatment for multiple myeloma (MM) has remarkably improved because of novel agents, such as bortezomib, thalidomide, and lenalidomide. [1][2][3] However, MM remains incurable, and nextgeneration novel agents are urgently needed. Because of high levels of endoplasmic reticulum (ER) stress and adaptation by the unfolded protein response (UPR), targeting signaling by the UPR and blocking this key survival pathway represent a new therapeutic strategy. In mammalian cells, protein folding is proportionally fine-tuned to the metabolic state of the cell within its microenvironment. Extracellular insults, such as low nutrients, hypoxia, and multiple drugs, result in the accumulation of misfolded proteins in the ER, thereby causing ER stress and initiating the UPR. 4 The UPR in turn increases the biosynthetic capacity and decreases the biosynthetic burden of the ER, to maintain cellular homeostasis. However, when the stress cannot be compensated by the UPR, cellular apoptosis occurs. 5 The UPR consists of 3 branches of signaling pathways, which initiate from 3 ER transmembrane proteins: inositol-requiring enzyme 1␣ (IRE1␣), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). In the resting state, these proteins are associated with molecular chaperone BiP/GRP78 in the ER. However, when unfolded proteins accumulate in the ER, BiP/GRP78 dissociates from them, thereby inducing UPR signaling. 6 In the UPR, IRE1␣ is activated by oligomerization and autophosphorylation, resulting in activation of its endoribonuclease to cleave and initiate splicing of the X-box binding protein 1 (XBP1) mRNA. A 26-nucleotide intron from XBP1 is removed by activated IRE1␣ endoribonuclease, resulting in a translational frame-shift to modify unspliced XBP1 (XBP1u: inactive) into spliced XBP1 (XBP1s: active). 7 XBP1 is a unique transcription factor that regulates genes responsible for ER-associated degradation (ERAD), such as EDEM, and those responsible for promoting protein folding, such as p58IPK and other ER chaperones. 8 Thus, IRE1␣-XBP1 pathway has a prosurvival role in the UPR. However, under conditions of prolonged and uncompensated stress, the UPR leads to cellular apoptosis, known as the terminal UPR. The proapoptotic transcription factor CHOP, also known as GADD153, is induced via PERK and ATF6 pathways. CHOP causes downregulation of BCL2, thereby leading to caspase-dependent apoptosis. 9 IRE1␣ also has a proapoptotic role: it binds TRAF2 and activates ASK1, which causes JNK activation, thereby leading to caspase-dependent apoptosis. 10 ...

Purpose PD1/PD-L1 signaling promotes tumor growth while inhibiting effector cell-mediated anti-tumor immune responses. Here we assessed the impact of single and dual blockade of PD1/PD-L1, alone or in combination with lenalidomide, on accessory and immune cell function as well as multiple myeloma (MM) cell growth in the BM milieu. Experimental Design Surface expression of PD1 on immune effector cells, and PD-L1 expression on CD138+MM cells and myeloid derived suppressor cells (MDSC) were determined on tumor cells from newly diagnosed (ND)-MM and relapsed/refractory (RR)-MM BM versus healthy donor (HD). We defined the impact of single and dual blockade of PD1/PD-L1, alone and with lenalidomide, on autologous anti-MM immune response and tumor cell growth. Results Both ND and RR patient MM cells have increased PD-L1 mRNA and surface expression compared to HD. There is also a significant increase in PD1 expression on effector cells in MM. Importantly, PD1/PD-L1-blockade abrogates BM-stroma cell (BMSC)-induced MM growth, and combined blockade of PD1/PD-L1 with lenalidomide further inhibits BMSC-induced tumor growth. These effects are associated with induction of intracellular expression of IFNγ and Granzyme-B in effector cells. Importantly, PD-L1 expression in MM is higher on MDSC than on antigen presenting cells, and PD1/PD-L1-blockade inhibits MDSC-mediated MM growth. Finally, lenalidomide with PD1/PD-L1-blockade inhibits MDSC-mediated immune suppression. Conclusion Our data therefore demonstrates that checkpoint signaling plays an important role in providing the tumor-promoting, immune-suppressive microenvironment in MM, and that PD1/PD-L1-blockade induces anti-MM immune response that can be enhanced by lenalidomide, providing the framework for clinical evaluation of combination therapy.

Histone deacetylases (HDACs) represent novel molecular targets for the treatment of various types of cancers, including multiple myeloma (MM). Many HDAC inhibitors have already shown remarkable anti-tumor activities in the preclinical setting; however, their clinical utility is limited due to unfavorable toxicities associated with their broad range HDAC inhibitory effects. Isoform-selective HDAC inhibition may allow for MM cytotoxicity without attendant side effects. In this study, we demonstrated that HDAC3 knockdown and a small molecule HDAC3 inhibitor BG45 trigger significant MM cell growth inhibition via apoptosis, evidenced by caspase and PARP cleavage. Importantly, HDAC3 inhibition downregulates phosphorylation (tyrosine 705 and serine 727) of STAT3. Neither IL-6 nor bone marrow stromal cells overcome this inhibitory effect of HDAC3 inhibition on p-STAT3 and MM cell growth. Moreover, HDAC3 inhibition also triggers hyperacetylation of STAT3, suggesting crosstalk signaling between phosphorylation and acetylation of STAT3. Importantly, inhibition of HDAC3, but not HDAC1 or HDAC2, significantly enhances bortezomib-induced cytotoxicity. Finally, we confirm that BG45 alone and in combination with bortezomib trigger significant tumor growth inhibition in vivo in a murine xenograft model of human MM. Our results indicate that HDAC3 represents a promising therapeutic target, and validate a prototype novel HDAC3 inhibitor BG45 in MM.