Megan C. Duggan

Myeloid-derived suppressor cells express Bruton's tyrosine kinase and can be depleted in tumor bearing hosts by ibrutinib treatment

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of immature myeloid cells that expand in tumor-bearing hosts in response to soluble factors produced by tumor and stromal cells. MDSC expansion has been linked to loss of immune effector cell function and reduced efficacy of immune-based cancer therapies, highlighting the MDSC population as an attractive therapeutic target. Ibrutinib, an irreversible inhibitor of Brutons tyrosine kinase (BTK) and IL2-inducible T-cell kinase (ITK), is in clinical use for the treatment of B-cell malignancies. Here, we report that BTK is expressed by murine and human MDSCs, and that ibrutinib is able to inhibit BTK phosphorylation in these cells. Treatment of MDSCs with ibrutinib significantly impaired nitric oxide production and cell migration. In addition, ibrutinib inhibited in vitro generation of human MDSCs and reduced mRNA expression of indolamine 2,3-dioxygenase, an immunosuppressive factor. Treatment of mice bearing EMT6 mammary tumors with ibrutinib resulted in reduced frequency of MDSCs in both the spleen and tumor. Ibrutinib treatment also resulted in a significant reduction of MDSCs in wild-type mice bearing B16F10 melanoma tumors, but not in X-linked immunodeficiency mice (XID) harboring a BTK mutation, suggesting that BTK inhibition plays an important role in the observed reduction of MDSCs in vivo Finally, ibrutinib significantly enhanced the efficacy of anti-PD-L1 (CD274) therapy in a murine breast cancer model. Together, these results demonstrate that ibrutinib modulates MDSC function and generation, revealing a potential strategy for enhancing immune-based therapies in solid malignancies. Cancer Res; 76(8); 2125-36. ©2016 AACR.

MicroRNA-3151 inactivates TP53 in BRAF-mutated human malignancies

Significance Activating mutations in the B-Raf proto-oncogene serine/threonine kinase (BRAF) gene occur in many tumor types, the highest incidence being in malignant melanoma and papillary thyroid carcinoma. In patients with BRAF mutations tumor progression is more rapid than in patients without these mutations. Therapeutic strategies presently aim at inhibiting BRAF resulting in slower tumor progression; however, lasting remission is rarely accomplished. In this paper we identify the oncomiR-3151 as a downstream effector of mutated BRAF. MicroRNA-3151 (miR-3151) targets TP53 and other members of the TP53 pathway resulting in its inhibition. Simultaneous inhibition of BRAF and miR-3151 potentiates the effects on tumor cell growth. These data establish a link between mutated BRAF and the TP53 pathway, allowing novel therapeutic approaches to be considered. The B-Raf proto-oncogene serine/threonine kinase (BRAF) gene is the most frequently mutated gene in malignant melanoma (MM) and papillary thyroid cancer (PTC) and is causally involved in malignant cell transformation. Mutated BRAF is associated with an aggressive disease phenotype, thus making it a top candidate for targeted treatment strategies in MM and PTC. We show that BRAF mutations in both MM and PTC drive increased expression of oncomiR-3151, which is coactivated by the SP1/NF-κB complex. Knockdown of microRNA-3151 (miR-3151) with short hairpin RNAs reduces cell proliferation and increases apoptosis of MM and PTC cells. Using a targeted RNA sequencing approach, we mechanistically determined that miR-3151 directly targets TP53 and other members of the TP53 pathway. Reducing miR-3151’s abundance increases TP53’s mRNA and protein expression and favors its nuclear localization. Consequently, knockdown of miR-3151 also leads to caspase-3–dependent apoptosis. Simultaneous inhibition of aberrantly activated BRAF and knockdown of miR-3151 potentiates the effects of sole BRAF inhibition with the BRAF inhibitor vemurafenib and may provide a novel targeted therapeutic approach in BRAF-mutated MM and PTC patients. In conclusion, we identify miR-3151 as a previously unidentified player in MM and PTC pathogenesis, which is driven by BRAF-dependent and BRAF-independent mechanisms. Characterization of TP53 as a downstream effector of miR-3151 provides evidence for a causal link between BRAF mutations and TP53 inactivation.

MICA-Expressing Monocytes Enhance Natural Killer Cell Fc Receptor-Mediated Antitumor Functions

Natural killer (NK) cells secrete immunostimulatory factors like IFNγ in response to tumors. Engagement of monocyte MICA and NK cell NKG2D promoted and enhanced the NK response to HER2+ breast tumors treated with mAb to HER2 in a murine model. Natural killer (NK) cells are large granular lymphocytes that promote the antitumor response via communication with other cell types in the tumor microenvironment. Previously, we have shown that NK cells secrete a profile of immune stimulatory factors (e.g., IFNγ, MIP-1α, and TNFα) in response to dual stimulation with the combination of antibody (Ab)-coated tumor cells and cytokines, such as IL12. We now demonstrate that this response is enhanced in the presence of autologous monocytes. Monocyte enhancement of NK cell activity was dependent on cell-to-cell contact as determined by a Transwell assay. It was hypothesized that NK cell effector functions against Ab-coated tumor cells were enhanced via binding of MICA on monocytes to NK cell NKG2D receptors. Strategies to block MICA–NKG2D interactions resulted in reductions in IFNγ production. Depletion of monocytes in vivo resulted in decreased IFNγ production by murine NK cells upon exposure to Ab-coated tumor cells. In mice receiving trastuzumab and IL12 therapy, monocyte depletion resulted in significantly greater tumor growth in comparison to mock-depleted controls (P < 0.05). These data suggest that NK cell–monocyte interactions enhance NK cell antitumor activity in the setting of monoclonal Ab therapy for cancer. Cancer Immunol Res; 5(9); 778–89. ©2017 AACR.

Nitric Oxide Production by Myeloid Derived Suppressor Cells Plays a Role in Impairing Fc Receptor-Mediated Natural Killer Cell Function.

Purpose: mAbs are used to treat solid and hematologic malignancies and work in part through Fc receptors (FcRs) on natural killer cells (NK). However, FcR-mediated functions of NK cells from patients with cancer are significantly impaired. Identifying the mechanisms of this dysfunction and impaired response to mAb therapy could lead to combination therapies and enhance mAb therapy. Experimental Design: Cocultures of autologous NK cells and MDSC from patients with cancer were used to study the effect of myeloid-derived suppressor cells (MDSCs) on NK-cell FcR-mediated functions including antibody-dependent cellular cytotoxicity, cytokine production, and signal transduction in vitro. Mouse breast cancer models were utilized to study the effect of MDSCs on antibody therapy in vivo and test the efficacy of combination therapies including a mAb and an MDSC-targeting agent. Results: MDSCs from patients with cancer were found to significantly inhibit NK-cell FcR-mediated functions including antibody-dependent cellular cytotoxicity, cytokine production, and signal transduction in a contact-independent manner. In addition, adoptive transfer of MDSCs abolished the efficacy of mAb therapy in a mouse model of pancreatic cancer. Inhibition of iNOS restored NK-cell functions and signal transduction. Finally, nonspecific elimination of MDSCs or inhibition of iNOS in vivo significantly improved the efficacy of mAb therapy in a mouse model of breast cancer. Conclusions: MDSCs antagonize NK-cell FcR-mediated function and signal transduction leading to impaired response to mAb therapy in part through nitric oxide production. Thus, elimination of MDSCs or inhibition of nitric oxide production offers a strategy to improve mAb therapy. Clin Cancer Res; 24(8); 1891–904. ©2018 AACR.

The combination of MLN2238 (ixazomib) with interferon-alpha results in enhanced cell death in melanoma

The ubiquitin-proteasome signaling pathway is critical for cell cycle regulation and neoplastic growth. Proteasome inhibition can activate apoptotic pathways. Bortezomib, a selective proteasome inhibitor, has anti-melanoma activity. MLN2238 (ixazomib), an oral proteasome inhibitor, has improved pharmacotherapeutic parameters compared to bortezomib. Interferon-alpha (IFN-α), an immune boosting agent, is FDA-approved for treatment of melanoma. In this study in vitro and in vivo evaluation of the antitumor potential of ixazomib and combination treatments with ixazomib and IFN-α were performed. Apoptosis induced by ixazomib was first observed at 12 hours and was maximal at 48 hours with similar levels of cell death compared to bortezomib. IFN-α alone had little effect on cell viability in vitro. However, the combination of ixazomib with IFN-α significantly enhanced ixazomibs ability to induce apoptotic cell death in BRAF V600E mutant and BRAF wild-type human melanoma tumor cells. The combination of ixazomib and IFN-α also enhanced inhibition of cell proliferation in BRAF V600E mutant melanoma tumor cells; however, this was not seen in BRAF wild-type cells. Ixazomib-induced apoptosis was associated with processing of the pro-apoptotic proteins procaspase-3, -7, -8, and -9, and cleavage of poly-ADP-ribose polymerase (PARP). In an in vivo xenograft model of human melanoma, combination treatment with IFN-α-2b and ixazomib demonstrated a significant reduction in tumor volume when compared to vehicle (p = 0.005) and single therapy ixazomib (p = 0.017) and IFN-α-2b (p = 0.036). These pre-clinical results support further evaluation of combination treatment with ixazomib and IFN-α for the treatment of advanced BRAF V600E mutant melanoma.

Identification of NRAS isoform 2 overexpression as a mechanism facilitating BRAF inhibitor resistance in malignant melanoma

Significance This study indicates that one of the isoforms of NRAS, specifically NRAS isoform 2, plays a role in BRAF inhibitor resistance by facilitating alternative survival signaling through the PI3K pathway in the presence of MAPK pathway inhibition. Targeting NRAS isoform 2 may be a beneficial treatment strategy in the prevention and management of BRAF inhibitor resistance in melanoma. Activating mutations in BRAF are found in 50% of melanomas and although treatment with BRAF inhibitors (BRAFi) is effective, resistance often develops. We now show that recently discovered NRAS isoform 2 is up-regulated in the setting of BRAF inhibitor resistance in melanoma, in both cell lines and patient tumor tissues. When isoform 2 was overexpressed in BRAF mutant melanoma cell lines, melanoma cell proliferation and in vivo tumor growth were significantly increased in the presence of BRAFi treatment. shRNA-mediated knockdown of isoform 2 in BRAFi resistant cells restored sensitivity to BRAFi compared with controls. Signaling analysis indicated decreased mitogen-activated protein kinase (MAPK) pathway signaling and increased phosphoinositol-3-kinase (PI3K) pathway signaling in isoform 2 overexpressing cells compared with isoform 1 overexpressing cells. Immunoprecipitation of isoform 2 validated a binding affinity of this isoform to both PI3K and BRAF/RAF1. The addition of an AKT inhibitor to BRAFi treatment resulted in a partial restoration of BRAFi sensitivity in cells expressing high levels of isoform 2. NRAS isoform 2 may contribute to resistance to BRAFi by facilitating PI3K pathway activation.

Abstract P4-09-18: Characterization of circulating myeloid derived suppressor cells and cytokines in patients undergoing neo-adjuvant chemotherapy for breast cancer

Myeloid derived suppressor cells (MDSC) are immature immune cells that expand in patients (pts) with cancer and suppress anti-tumor immunity. MDSC are also known to support angiogenesis. Higher circulating MDSC levels are seen in patients with greater tumor burden. Therefore, circulating MDSC levels could be affected by chemotherapy and could correlate with response. In this prospective pilot trial, peripheral blood (PB) levels of granulocytic (G-MDSC) and monocytic (M-MDSC) MDSC were measured in pts with operable breast cancer (BC) treated with neo-adjuvant chemotherapy (NAC) to study their association with pathologic complete response. It was hypothesized that MDSC % would show an association with complete pathologic response (pCR). The association of 10 different cytokine levels (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IFN-γ, TNF-α) with pCR was also explored. Linear mixed models tested the associations between MDSC % or cytokines across time points with pCR. Levels of MDSC were measured by flow cytometry as a % of PB mononuclear cells prior to cycle (C) 1 and 2 of doxorubicin and cyclophosphamide (AC) and 1st and last administration of paclitaxel (T) or T and anti-HER2 therapy (in HER2+ pts). For other regimens, MDSC were measured prior to 1st, 2nd and last cycle. MDSC were defined as HLA-DR-, CD11b+, CD33+ cells with G-MDSC and M-MDSC cells expressing CD15 and CD14, respectively. Plasma cytokine levels were measured using a multiplex assay (Bio-Rad). Of 24 enrolled pts, 1, 20 and 3 had clinical stage I, II, IIIA, respectively. Median age was 48 (range 32-70). 11, 6 and 7 pts were triple negative (TN), HER2+ and hormone receptor (HR)+, respectively. PCR rate was 45.8% (46%, 50%, 43%, 20% for TN, HER2+, HR+ and >10% HR+ pts). Rate of residual cancer burden (RCB) class 0-1 was 58.3% (63.6%, 50%, 57.1%, 40% in TN, HER2+, HR+ and >10% HR+ pts). Mean M-MDSC % were Citation Format: Wesolowski R, Duggan M, Stiff A, Trikha P, Schoenfield L, Abdel-Rasoul M, Layman R, Ramaswamy B, Macrae E, Lustberg MB, Mrozek E, Carson WE. Characterization of circulating myeloid derived suppressor cells and cytokines in patients undergoing neo-adjuvant chemotherapy for breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-18.

Modeling combination therapy for breast cancer with BET and immune checkpoint inhibitors

Significance The Bromo- and Extra-Terminal (BET) family proteins regulate transcription of several oncogenes. For this reason, targeting BET protein may be a promising strategy for cancer therapy. Checkpoint inhibitors, such as anti–CTLA-4, sustain cytotoxic T cells in their anticancer activity. This paper develops a mathematical model to determine the efficacy of combination therapy with BET and CTLA-4 inhibitors. Simulations of the model are in agreement with experimental results. It is shown that the two drugs are positively correlated in reducing the tumor volume. The model also shows, under different combinations of doses that lead to the same tumor volume reduction, in what proportion to choose the two drugs to minimize the increased level of the proinflammatory TNF-α. CTLA-4 is an immune checkpoint expressed on active anticancer T cells. When it combines with its ligand B7 on dendritic cells, it inhibits the activity of the T cells. The Bromo- and Extra-Terminal (BET) protein family includes proteins that regulate the expression of key oncogenes and antiapoptotic proteins. BET inhibitor (BETi) has been shown to reduce the expression of MYC by suppressing its transcription factors and to down-regulate the hypoxic transcriptome response to VEGF-A. This paper develops a mathematical model of the treatment of cancer by combination therapy of BETi and CTLA-4 inhibitor. The model shows that the two drugs are positively correlated in the sense that the tumor volume decreases as the dose of each of the drugs is increased. The model also considers the effect of the combined therapy on levels of myeloid-derived suppressor cells (MDSCs) and the overexpression of TNF-α, which may predict gastrointestinal side effects of the combination.

Co-stimulation of the fc receptor and interleukin-12 receptor on human natural killer cells leads to increased expression of cd25

ABSTRACT Natural killer (NK) cells serve a critical role in the immune response against microbes and developing tumors. We have demonstrated that NK cells produce stimulatory cytokines (e.g., IFN-γ) in response to potent stimulation via immobilized IgG (to engage Fc receptors) and interleukin (IL)-12. CD25 is a component of the high-affinity IL-2R, which promotes NK cell activation in response to low doses of IL-2 such as those released by activated T cells. We hypothesized that stimulation of NK cells via IgG and IL-12 would enhance CD25 expression and promote NK cell anti-tumor activity in response to low-dose IL-2. It was confirmed that this dual stimulation strategy significantly enhanced NK cell CD25 expression compared to unstimulated cells or cells treated with IgG or IL-12 alone. Dual stimulated NK cells also were more responsive to low-dose IL-2. Dual stimulated NK cells subsequently treated with low-dose IL-2 (10 pg/mL) displayed enhanced intracellular signaling as indicated by increased pSTAT5 levels. IFN-γ production and cytotoxicity against K562 cells by NK cells stimulated with low-dose IL-2 was comparable to that of cells treated with high-dose IL-2 (10 ng/mL). Importantly, cells isolated from head and neck cancer patients receiving the mAb cetuximab and IL-12 on a clinical trial displayed increased CD25 expression following combination therapy compared to baseline. Altogether, these findings suggest that FcR and IL-12R co-stimulation induces expression of the high-affinity IL-2R and promotes NK cell anti-tumor activity.

Activation of the FcgammaReceptorIIIa on human natural killer cells leads to increased expression of functional interleukin-21 receptor

ABSTRACT Natural killer (NK) cells are innate immune effector cells that play a crucial role in immune surveillance and the destruction of cancer cells. NK cells express a low-affinity receptor for the Fc or constant region of immunoglobulin G (FcγRIIIa) and multiple cytokine receptors that respond to antibody-coated targets and cytokines in the tumor microenvironment. In the present work, microarray gene expression analysis revealed that the IL-21 receptor (IL-21R) was strongly upregulated following FcR stimulation. The IL-21R was found to be upregulated on FcR-stimulated NK cells at the transcript level as determined by reverse transcription polymerase chain reaction (RT-PCR). Immunoblot analysis revealed that protein expression of the IL-21R peaked at 8 h post-stimulation of the FcR. Inhibition of the mitogen-activated protein kinase (MAPK) pathway downstream of the FcR blocked the induction of IL-21R expression. Increased expression of the IL-21R sensitized NK cells to IL-21 stimulation, as treatment of FcR-stimulated NK cells led to significantly increased phosphorylation of STAT1 and STAT3, as measured by intracellular flow cytometry and immunoblot analysis. Following FcR-stimulation, IL-21-activated NK cells were better able to mediate the lysis of trastuzumab-coated human epidermal growth factor receptor 2 (HER2+) SK-BR-3 tumor cells as compared to control-treated cells. Likewise, IL-21-induced NK cell secretion of IFNγ following exposure to antibody-coated tumor cells was enhanced following FcR-stimulation. The analysis of NK cells from patients receiving trastuzumab therapy for HER2+ cancer exhibited increased levels of the IL-21R following the administration of antibody suggesting that the presence of monoclonal antibody-coated tumor cells in vivo can stimulate the increased expression of IL-21R on NK cells.