PD-1/PD-L1 signaling promotes tumor growth while inhibiting effector cell-mediated antitumor immune responses. Here, we assessed the impact of single and dual blockade of PD-1/PD-L1, alone or in ...combination with lenalidomide, on accessory and immune cell function as well as multiple myeloma cell growth in the bone marrow (BM) milieu.
Surface expression of PD-1 on immune effector cells, and PD-L1 expression on CD138(+) multiple myeloma cells and myeloid-derived suppressor cells (MDSC) were determined in BM from newly diagnosed (ND) multiple myeloma and relapsed/refractory (RR) multiple myeloma versus healthy donor (HD). We defined the impact of single and dual blockade of PD-1/PD-L1, alone and with lenalidomide, on autologous anti-multiple myeloma immune response and tumor cell growth.
Both ND and RR patient multiple myeloma cells have increased PD-L1 mRNA and surface expression compared with HD. There is also a significant increase in PD-1 expression on effector cells in multiple myeloma. Importantly, PD-1/PD-L1 blockade abrogates BM stromal cell (BMSC)-induced multiple myeloma growth, and combined blockade of PD-1/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 multiple myeloma is higher on MDSC than on antigen-presenting cells, and PD-1/PD-L1 blockade inhibits MDSC-mediated multiple myeloma growth. Finally, lenalidomide with PD-1/PD-L1 blockade inhibits MDSC-mediated immune suppression.
Our data therefore demonstrate that checkpoint signaling plays an important role in providing the tumor-promoting, immune-suppressive microenvironment in multiple myeloma, and that PD-1/PD-L1 blockade induces anti-multiple myeloma immune response that can be enhanced by lenalidomide, providing the framework for clinical evaluation of combination therapy.
Summary
CXCR4WHIM somatic mutations are distinctive to Waldenström Macroglobulinaemia (WM), and impact disease presentation and treatment outcome. The clonal architecture of CXCR4WHIM mutations ...remains to be delineated. We developed highly sensitive allele‐specific polymerase chain reaction (AS‐PCR) assays for detecting the most common CXCR4WHIM mutations (CXCR4S338X C>A and C>G) in WM. The AS‐PCR assays detected CXCR4S338X mutations in WM and IgM monoclonal gammopathy of unknown significance (MGUS) patients not revealed by Sanger sequencing. By combined AS‐PCR and Sanger sequencing, CXCR4WHIM mutations were identified in 44/102 (43%), 21/62 (34%), 2/12 (17%) and 1/20 (5%) untreated WM, previously treated WM, IgM MGUS and marginal zone lymphoma patients, respectively, but no chronic lymphocytic leukaemia, multiple myeloma, non‐IgM MGUS patients or healthy donors. Cancer cell fraction analysis in WM and IgM MGUS patients showed CXCR4S338X mutations were primarily subclonal, with highly variable clonal distribution (median 35·1%, range 1·2–97·5%). Combined AS‐PCR and Sanger sequencing revealed multiple CXCR4WHIM mutations in many individual WM patients, including homozygous and compound heterozygous mutations validated by deep RNA sequencing. The findings show that CXCR4WHIM mutations are more common in WM than previously revealed, and are primarily subclonal, supporting their acquisition after MYD88L265P in WM oncogenesis. The presence of multiple CXCR4WHIM mutations within individual WM patients may be indicative of targeted CXCR4 genomic instability.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Myeloid differentiation factor 88 (MYD88) L265P somatic mutation is highly prevalent in Waldenström macroglobulinemia (WM) and supports malignant growth through nuclear factor κB (NF-κB). The ...signaling cascade(s) by which MYD88 L265P promotes NF-κB activation in WM remain unclear. By lentiviral knockdown or use of a MYD88 inhibitor, decreased phosphorylation of the NF-κB gatekeeper IκBα and survival occurred in MYD88 L265P-expressing WM cells. Conversely, WM cells engineered to overexpress MYD88 L265P showed enhanced survival. Coimmunoprecipitation studies identified Bruton tyrosine kinase (BTK) complexed to MYD88 in L265P-expressing WM cells, with preferential binding of MYD88 to phosphorylated BTK (pBTK). Increased pBTK was also observed in WM cells transduced to overexpress L265P vs wild-type MYD88. Importantly, MYD88 binding to BTK was abrogated following treatment of MYD88 L265P-expressing cells with a BTK kinase inhibitor. Inhibition of BTK or interleukin-1 receptor-associated kinase 1 and 4 (IRAK-1 and -4) kinase activity induced apoptosis of WM cells, and their combination resulted in more robust inhibition of NF-κB signaling and synergistic WM cell killing. The results establish BTK as a downstream target of MYD88 L265P signaling, and provide a framework for the study of BTK inhibitors alone, and in combination with IRAK inhibitors for the treatment of WM.
•MYD88 L265P is a widely expressed somatic mutation in WM patients that supports NF-κB signaling through stimulation of BTK and IRAK 1/4.•Combined suppression of BTK and IRAK in MYD88 L265P expressing WM cells promotes synergistic inhibition of NF-κB signaling and WM cell killing.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cytotoxic chemotherapy targets elements common to all nucleated human cells, such as DNA and microtubules, yet it selectively kills tumor cells. Here we show that clinical response to these drugs ...correlates with, and may be partially governed by, the pretreatment proximity of tumor cell mitochondria to the apoptotic threshold, a property called mitochondrial priming. We used BH3 profiling to measure priming in tumor cells from patients with multiple myeloma, acute myelogenous and lymphoblastic leukemia, and ovarian cancer. This assay measures mitochondrial response to peptides derived from proapoptotic BH3 domains of proteins critical for death signaling to mitochondria. Patients with highly primed cancers exhibited superior clinical response to chemotherapy. In contrast, chemoresistant cancers and normal tissues were poorly primed. Manipulation of mitochondrial priming might enhance the efficacy of cytotoxic agents.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Summary
Lenalidomide (LEN) acts directly on multiple myeloma (MM) cells by inducing cereblon‐mediated degradation of interferon regulatory factor 4, Ikaros (IKZF)1 and IKZF3, transcription factors ...that are essential for MM cell survival. The mucin 1 (MUC1) C‐terminal transmembrane subunit (MUC1‐C) oncoprotein is aberrantly expressed by MM cells and protects against reactive oxygen species (ROS)‐mediated MM cell death. The present studies demonstrate that targeting MUC1‐C with GO‐203, a cell‐penetrating peptide inhibitor of MUC1‐C homodimerization, is more than additive with LEN in downregulating the WNT/β‐catenin pathway, suppressing MYC, and inducing late apoptosis/necrosis. We show that the GO‐203/LEN combination acts by synergistically increasing ROS and, in turn, suppressing β‐catenin. LEN resistance has been linked to activation of the WNT/β‐catenin→CD44 pathway. In this regard, our results further demonstrate that targeting MUC1‐C is effective against LEN‐resistant MM cells. Moreover, GO‐203 resensitized LEN‐resistant MM cells to LEN treatment in association with suppression of β‐catenin and CD44. Targeting MUC1‐C also resulted in downregulation of CD44 on the surface of primary MM cells. These findings, and the demonstration that expression of MUC1 and CD44 significantly correlate in microarrays from primary MM cells, provide support for combining GO‐203 with LEN in the treatment of MM and in LEN‐resistance.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Immunomodulatory drugs and monoclonal antibody-based immunotherapies have significantly improved the prognosis of the patients with multiple myeloma (MM) in the recent years. These new classes of ...reagents target malignant plasma cells (PCs) and further modulate the immune microenvironment, which prolongs anti-MM responses and may prevent tumor occurrence. Since MM remains an incurable cancer for most patients, there continues to be a need to identify new tumor target molecules and investigate alternative cellular approaches using gene therapeutic strategies and novel treatment mechanisms. Osteoclasts (OCs), as critical multi-nucleated large cells responsible for bone destruction in >80% MM patients, have become an attractive cellular target for the development of novel MM immunotherapies. In MM, OCs are induced and activated by malignant PCs in a reciprocal manner, leading to osteolytic bone disease commonly associated with this malignancy. Significantly, bidirectional interactions between OCs and MM cells create a positive feedback loop to promote MM cell progression, increase angiogenesis, and inhibit immune surveillance
both cell-cell contact and abnormal production of multiple cytokines/chemokines. Most recently, hyper-activated OCs have been associated with activation of programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway, which impairs T cell proliferation and cytotoxicity against MM cells. Importantly, therapeutic anti-CD38 monoclonal antibodies and checkpoint inhibitors can alleviate OC-induced immune suppression. Furthermore, a proliferation-inducing ligand, abundantly secreted by OCs and OC precursors, significantly upregulates PD-L1 expression on MM cells, in addition to directly promoting MM cell proliferation and survival. Coupled with increased PD-L1 expression in other immune-suppressive cells, i.e., myeloid-derived suppressor cells and tumor-associated macrophages, these results strongly suggest that OCs contribute to the immunosuppressive MM BM microenvironment. Based on these findings and ongoing osteoimmunology studies, therapeutic interventions targeting OC number and function are under development to diminish both MM bone disease and related immune suppression. In this review, we discuss the classical and novel roles of OCs in the patho-immunology of MM. We also describe novel therapeutic strategies simultaneously targeting OCs and MM interactions, including PD-1/PD-L1 axis, to overcome the immune-suppressive microenvironment and improve patient outcome.
Neuroblastoma is the most common malignant disease of infancy, and amplification of the
oncogene is closely associated with poor prognosis. Recently, expression of MYCN was shown to be inversely ...correlated with aryl hydrocarbon receptor (AHR) expression in neuroblastoma, and overexpression of AHR downregulated MYCN expression, promoting cell differentiation. Therefore, we further investigated the potential of AHR to serve as a prognostic indicator or a therapeutic target in neuroblastoma. First, the clinical significance of AHR in neuroblastoma was examined. Positive AHR immunostaining strongly correlated with differentiated histology of neuroblastoma and predicted better survival for patients. The mouse xenograft model showed that overexpression of AHR significantly suppressed neuroblastoma tumor growth. In addition, activation of AHR by the endogenous ligand kynurenine inhibited cell proliferation and promoted cell differentiation
and
. kynurenine treatment also upregulated the expression of
, a tumor metastasis suppressor, and attenuated metastasis in the xenograft model. Finally, analysis of
levels in neuroblastoma patient tumors using the R2: Genomics Analysis and Visualization Platform revealed that
expression positively correlated with
, and high
expression predicted better survival for patients. In conclusion, our results indicate that AHR is a novel prognostic biomarker for neuroblastoma, and that overexpression or activation of AHR offers a new therapeutic possibility for patients with neuroblastoma. SIGNIFICANCE: These findings show that AHR may function as a tumor suppressor in childhood neuroblastoma, potentially influencing the aetiologic and therapeutic targeting of the disease.
By whole-genome and/or Sanger sequencing, we recently identified a somatic mutation (MYD88 L265P) that stimulates nuclear factor κB activity and is present in >90% of Waldenström macroglobulinemia ...(WM) patients. MYD88 L265P was absent in 90% of immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS) patients. We therefore developed conventional and real-time allele-specific polymerase chain reaction (AS-PCR) assays for more sensitive detection and quantification of MYD88 L265P. Using either assay, MYD88 L265P was detected in 97 of 104 (93%) WM and 13 of 24 (54%) IgM MGUS patients and was either absent or rarely expressed in samples from splenic marginal zone lymphoma (2/20; 10%), CLL (1/26; 4%), multiple myeloma (including IgM cases, 0/14), and immunoglobulin G MGUS (0/9) patients as well as healthy donors (0/40; P < 1.5 × 10−5 for WM vs other cohorts). Real-time AS-PCR identified IgM MGUS patients progressing to WM and showed a high rate of concordance between MYD88 L265P ΔCT and BM disease involvement (r = 0.89, P = .008) in WM patients undergoing treatment. These studies identify MYD88 L265P as a widely present mutation in WM and IgM MGUS patients using highly sensitive and specific AS-PCR assays with potential use in diagnostic discrimination and/or response assessment. The finding of this mutation in many IgM MGUS patients suggests that MYD88 L265P may be an early oncogenic event in WM pathogenesis.
•MYD88 L265P is expressed in WM and IgM MGUS patients using AS-PCR assays with potential use in diagnostic discrimination and response assessment.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Despite significant advances in the treatment of multiple myeloma (MM), most patients succumb to disease progression. One of the major immunosuppressive mechanisms that is believed to play a role in ...myeloma progression is the expansion of regulatory T cells (Tregs). In this study, we demonstrate that myeloma cells drive Treg expansion and activation by secreting type 1 interferon (IFN). Blocking IFN α and β receptor 1 (IFNAR1) on Tregs significantly decreases both myeloma-associated Treg immunosuppressive function and myeloma progression. Using syngeneic transplantable murine myeloma models and bone marrow (BM) aspirates of MM patients, we found that Tregs were expanded and activated in the BM microenvironment at early stages of myeloma development. Selective depletion of Tregs led to a complete remission and prolonged survival in mice injected with myeloma cells. Further analysis of the interaction between myeloma cells and Tregs using gene sequencing and enrichment analysis uncovered a feedback loop, wherein myeloma-cell-secreted type 1 IFN induced proliferation and expansion of Tregs. By using IFNAR1-blocking antibody treatment and IFNAR1-knockout Tregs, we demonstrated a significant decrease in myeloma-associated Treg proliferation, which was associated with longer survival of myeloma-injected mice. Our results thus suggest that blocking type 1 IFN signaling represents a potential strategy to target immunosuppressive Treg function in MM.
The number and activity of osteoclasts (OCs) are strongly enhanced by myeloma cells, leading to significant bone lesions in patients with multiple myeloma (MM). Mechanisms remain elusive as to ...whether myeloma-supporting OCs also induce suppressive immune bone marrow (BM) microenvironment. Here, we first show that OCs significantly protect MM cells against T-cell–mediated cytotoxicity via direct inhibition of proliferating CD4+ and CD8+ T cells. The immune checkpoint molecules programmed death ligand 1 (PD-L1), Galectin-9, herpesvirus entry mediator (HVEM), and CD200, as well as T-cell metabolism regulators indoleamine 2, 3-dioxygenase (IDO), and CD38 are significantly upregulated during osteoclastogenesis. Importantly, the levels of these molecules, except CD38, are higher in OCs than in MM cells. Anti–PD-L1 monoclonal antibody (mAb) and IDO inhibitor partly overcome OC-inhibited T-cell responses against MM cells, confirming their roles in OC-suppressed MM cell lysis by cytotoxic T cells. In addition, Galectin-9 and a proliferation-induced ligand (APRIL), secreted by OCs, are significantly upregulated during osteoclastogenesis. Galectin-9 specifically induces apoptosis of T cells while sparing monocytes and MM cells. APRIL induces PD-L1 expression in MM cells, providing additional immune inhibition by OCs. Moreover, CD38 is significantly upregulated during osteoclastogenesis. When targeted by an anti-CD38 mAb, suppressive T-cell function by OCs is alleviated, associated with downregulation of HVEM and IDO. Taken together, these results define the expression of multiple immune proteins and cytokines in OCs essential for suppressive MM BM milieu. These results further support the combination of targeting these molecules to improve anti-MM immunity.
•OCs play a crucial role in myeloma-induced immunosuppressive microenvironment.•Therapeutic anti-CD38 mAb partially overcomes the immunosuppressive effect of OCs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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