Jennifer Yang

PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27Kip1

Protein arginine methyltransferase-5 (PRMT5) is a Type II arginine methyltransferase that regulates various cellular functions. We hypothesized that PRMT5 plays a role in regulating the growth of human melanoma cells. Immunohistochemical analysis indicated significant upregulation of PRMT5 in human melanocytic nevi, malignant melanomas and metastatic melanomas as compared to normal epidermis. Furthermore, nuclear PRMT5 was significantly decreased in metastatic melanomas as compared to primary cutaneous melanomas. In human metastatic melanoma cell lines, PRMT5 was predominantly cytoplasmic, and associated with its enzymatic cofactor Mep50, but not STAT3 or cyclin D1. However, histologic examination of tumor xenografts from athymic mice revealed heterogeneous nuclear and cytoplasmic PRMT5 expression. Depletion of PRMT5 via siRNA inhibited proliferation in a subset of melanoma cell lines, while it accelerated growth of others. Loss of PRMT5 also led to reduced expression of MITF (microphthalmia-associated transcription factor), a melanocyte-lineage specific oncogene, and increased expression of the cell cycle regulator p27Kip1. These results are the first to report elevated PRMT5 expression in human melanoma specimens and indicate this protein may regulate MITF and p27Kip1 expression in human melanoma cells.

Novel Small Molecule XPO1/CRM1 Inhibitors Induce Nuclear Accumulation of TP53, Phosphorylated MAPK and Apoptosis in Human Melanoma Cells

XPO1/CRM1 is a key nuclear exporter protein that mediates translocation of numerous cellular regulatory proteins. We investigated whether XPO1 is a potential therapeutic target in melanoma using novel selective inhibitors of nuclear export (SINE). In vitro effects of SINE on cell growth and apoptosis were measured by MTS assay and flow cytometry [Annexin V/propidium iodide (PI)], respectively in human metastatic melanoma cell lines. Immunoblot analysis was used to measure nuclear localization of key cellular proteins. The in vivo activity of oral SINE was evaluated in NOD/SCID mice bearing A375 or CHL-1 human melanoma xenografts. SINE compounds induced cytostatic and pro-apoptotic effects in both BRAF wild type and mutant (V600E) cell lines at nanomolar concentrations. The cytostatic and pro-apoptotic effects of XPO1 inhibition were associated with nuclear accumulation of TP53, and CDKN1A induction in the A375 cell line with wild type TP53, while pMAPK accumulated in the nucleus regardless of TP53 status. The orally bioavailable KPT-276 and KPT-330 compounds significantly inhibited growth of A375 (p<0.0001) and CHL-1 (p = 0.0087) human melanoma cell lines in vivo at well tolerated doses. Inhibition of XPO1 using SINE represents a potential therapeutic approach for melanoma across cells with diverse molecular phenotypes by promoting growth inhibition and apoptosis.

Chemogenetic Evaluation of the Mitotic Kinesin CENP-E Reveals a Critical Role in Triple-Negative Breast Cancer

Breast cancer patients with tumors lacking the three diagnostic markers (ER, PR, and HER2) are classified as triple-negative (primarily basal-like) and have poor prognosis because there is no disease-specific therapy available. To address this unmet medical need, gene expression analyses using more than a thousand breast cancer samples were conducted, which identified elevated centromere protein E (CENP-E) expression in the basal-a molecular subtype relative to other subtypes. CENP-E, a mitotic kinesin component of the spindle assembly checkpoint, is shown to be induced in basal-a tumor cell lines by the mitotic spindle inhibitor drug docetaxel. CENP-E knockdown by inducible shRNA reduces basal-a breast cancer cell viability. A potent, selective CENP-E inhibitor (PF-2771) was used to define the contribution of CENP-E motor function to basal-like breast cancer. Mechanistic evaluation of PF-2771 in basal-a tumor cells links CENP-E–dependent molecular events (e.g., phosphorylation of histone H3 Ser-10; phospho-HH3-Ser10) to functional outcomes (e.g., chromosomal congression defects). Across a diverse panel of breast cell lines, CENP-E inhibition by PF-2771 selectively inhibits proliferation of basal breast cancer cell lines relative to premalignant ones and its response correlates with the degree of chromosomal instability. Pharmacokinetic–pharmacodynamic efficacy analysis in a basal-a xenograft tumor model shows that PF-2771 exposure is well correlated with increased phospho-HH3-Ser10 levels and tumor growth regression. Complete tumor regression is observed in a patient-derived, basal-a breast cancer xenograft tumor model treated with PF-2771. Tumor regression is also observed with PF-2771 in a taxane-resistant basal-a model. Taken together, CENP-E may be an effective therapeutic target for patients with triple-negative/basal-a breast cancer. Mol Cancer Ther; 13(8); 2104–15. ©2014 AACR.

Dual Inhibition of MEK and PI3K/Akt Rescues Cancer Cachexia through Both Tumor Extrinsic and Intrinsic Activities.

Involuntary weight loss, a part of the cachexia syndrome, is a debilitating comorbidity of cancer and currently has no treatment options. Results from a recent clinical trial at our institution showed that biliary tract cancer patients treated with a MEK inhibitor exhibited poor tumor responses but surprisingly gained weight and increased their skeletal muscle mass. This implied that MEK inhibition might be anticachectic. To test this potential effect of MEK inhibition, we utilized the established Colon-26 model of cancer cachexia and the MEK1/2 inhibitor MEK162. Results showed that MEK inhibition effectively prevented muscle wasting. Importantly, MEK162 retained its ability to spare muscle loss even in mice bearing a Colon-26 clone resistant to the MEK inhibitor, demonstrating that the effects of blocking MEK are at least in part independent of the tumor. Because single-agent MEK inhibitors have been limited as a first-line targeted therapy due to compensatory activation of other oncogenic signaling pathways, we combined MEK162 with the PI3K/Akt inhibitor buparlisib. Results showed that this combinatorial treatment significantly reduced tumor growth due to a direct activity on Colon-26 tumor cells in vitro and in vivo, while also preserving skeletal muscle mass. Together, our results suggest that as a monotherapy, MEK inhibition preserves muscle mass, but when combined with a PI3K/Akt inhibitor exhibits potent antitumor activity. Thus, combinatorial therapy might serve as a new approach for the treatment of cancer cachexia. Mol Cancer Ther; 16(2); 344–56. ©2016 AACR. See related article by Kobayashi et al., p. 357

Single agent BMS-911543 Jak2 inhibitor has distinct inhibitory effects on STAT5 signaling in genetically engineered mice with pancreatic cancer

The Jak/STAT pathway is activated in human pancreatic ductal adenocarcinoma (PDAC) and cooperates with mutant Kras to drive initiation and progression of PDAC in murine models. We hypothesized that the small-molecule Jak2 inhibitor (BMS-911543) would elicit anti-tumor activity against PDAC and decrease immune suppressive features of the disease. We used an aggressive genetically engineered PDAC model with mutant KrasG12D, tp53R270H, and Brca1 alleles (KPC-Brca1 mice). Mice with confirmed tumor burden were treated orally with vehicle or 30 mg/kg BMS-911543 daily for 14 days. Histologic analysis of pancreata from treated mice revealed fewer foci of adenocarcinoma and significantly decreased Ki67+ cells versus controls. In vivo administration of BMS-911543 significantly reduced pSTAT5 and FoxP3 positive cells within the pancreas, but did not alter STAT3 phosphorylation. Continuous dosing of KPC-Brca1 mice with BMS-911543 resulted in a median survival of 108 days, as compared to a median survival of 87 days in vehicle treated animals, a 23% increase (p = 0.055). In vitro experiments demonstrated that PDAC cell lines were poorly sensitive to BMS-911543, requiring high micromolar concentrations to achieve targeted inhibition of Jak/STAT signaling. Similarly, BMS-911543 had little in vitro effect on the viability of both murine and human PDAC-derived stellate cell lines. However, BMS-911543 potently inhibited phosphorylation of pSTAT3 and pSTAT5 at low micromolar doses in human PBMC and reduced in vitro differentiation of Foxp3+ T regulatory cells. These results indicate that single agent Jak2i deserves further study in preclinical models of PDAC and has distinct inhibitory effects on STAT5 mediated signaling.

Signaling pathways as therapeutic targets in biliary tract cancer

ABSTRACT Introduction: The incidence of biliary tract cancer (BTC) is increasing, and the disease is frequently diagnosed during advanced stages, leading to poor overall survival. Limited treatment options are currently available and novel therapeutic approaches are needed. A number of completed clinical trials have evaluated the role of chemotherapy for BTC, demonstrating a marginal benefit. Thus, there is increased interest in applying targeted therapies for this disease. Areas covered: This review article summarizes the role of chemotherapeutic regimens for the treatment of BTC, and highlights key signal transduction pathways of interest for targeted inhibition. Of particular interest are the MEK or MAP2K (mitogen-activated protein kinase kinase), phosphatidylinositol-3 kinase (PI3K) and signal transducer and activator of transcription-3 (STAT3) pathways. We discuss the available data on several promising inhibitors of these pathways, both in the pre-clinical and clinical settings. Expert opinion: Future treatment strategies should address targeting of MEK, PI3K and STAT3 for BTC, with a focus on combined therapeutic approaches.

Selinexor, a selective inhibitor of nuclear export (SINE), shows enhanced activity in combination with PD-1/PD-L1 blockade in syngeneic murine models of colon cancer and melanoma

Exportin-1 (XPO1) is a nuclear export protein with >220 cargo proteins, including tumor suppressors and cell cycle modulators. Selinexor is a SINE (Selective Inhibitor of Nuclear Export) compound that has been administered to >900 cancer patients in Phase I and II trials to date, with evidence of efficacy and tolerability. Selinexor blocks nuclear export of NFAT1c, STAT1 and STAT3, which are implicated in regulating the inhibitory T cell receptor PD-1 and its ligand, PD-L1. We hypothesized that selinexor would upregulate T cell checkpoint molecule expression, and that combination treatment with anti-PD-1 or anti-PD-L1 would thereby enhance the ability of selinexor to elicit antitumor activity. Selinexor increased PD-1 gene expression by ~2-fold in normal lymphocytes and induced PD-L1 gene expression in tumor cell lines. Mice bearing syngeneic colon tumors (colon26) treated with selinexor and anti-PD-1 for 2 weeks demonstrated a significant reduction in tumor growth rate (P < 0.05), while monotherapy with either agent had no significant effect on tumor growth. Similar results were obtained in mice bearing syngeneic B16F10 melanoma tumors, whereby combined treatment with selinexor + anti-PD-1 was superior to either single agent alone (p < 0.034). Combined therapy of mice bearing B16F10 tumors with selinexor and anti-PD-L1 was similarly effective, with significantly smaller tumors at the study endpoint (p < 0.001). No weight loss or signs of toxicity were evident in any in vivo study. Immunophenotypic analysis by flow cytometry revealed that selinexor alone or in combination with anti-PD-1/anti-PD-L1 significantly increased the percentage of splenic NK cells (p≤0.050), while selinexor ± anti-PD-L1 significantly increased the percentage of splenic Th1 T cells (p≤0.011), all compared to vehicle treated mice. Interestingly, combining selinexor with anti-PD-L1 significantly decreased the percentage of splenocytes that expressed PD-L1 (p < 0.001). These data indicate that the efficacy of selinexor may be enhanced by disrupting the pre-existing PD-1/PD-L1 signaling in effector cells (T and NK cells). Altogether, these data suggest that the efficacy of selinexor in combination with anti-PD-1 or anti PD L1 in mouse syngeneic tumor models may be due to both disrupting immunosuppressive PD-1/PD-L1 signaling and increasing the frequency of potentially tumor reactive NK cells and Th1 T cells. This provides a rational basis for this treatment combination as a novel therapeutic approach for advanced cancer.

Abstract LB-254: PRMT5 is upregulated in malignant and metastatic melanoma, and regulates expression of the MITF transcription factor

Protein arginine methyltransferase 5 (PRMT5) is an enzyme which catalyzes the covalent attachment of methyl groups to arginine residues of various proteins. PRMT5 binds and/or methylates both nuclear (e.g. histones) and cytoplasmic (e.g. p53, CRAF) proteins to regulate cellular functions including gene expression, cell cycle, and apoptosis among others. PRMT5 has been characterized in leukemia, lymphoma, glioma, and breast cancer; however little is known regarding its role in malignant melanoma. We hypothesized that PRMT5 plays a unique role in regulating melanoma cell biology. PRMT5 expression was measured by IHC analysis of formalin-fixed samples from patients with melanoma (n=133 primary; n=66 metastases), benign nevi (n=24), and normal adjacent epidermis (n=21). PRMT5 expression was significantly elevated in melanoma samples compared to normal adjacent epidermis (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-254. doi:1538-7445.AM2012-LB-254

Abstract 2319: Selinexor, a selective inhibitor of nuclear export (SINE), enhances the in vivo efficacy of checkpoint blockade with antibodies targeting CTLA4 or PD-1/PD-L1 in melanoma

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Selinexor is a SINE (Selective Inhibitor of Nuclear Export) compound that has been administered to >1000 cancer patients in Phase I and II trials to date, with evidence of efficacy and tolerability. This small molecule targets exportin-1 (XPO1), a key nuclear export protein with >200 cargo proteins which include both tumor suppressors and cell cycle modulators. As a result, selinexor blocks nuclear export of proteins including IκB, NFAT1c, STAT1 and STAT3, which regulate expression of the inhibitory T cell receptors CTLA4, PD1 and its ligand, PD-L1. We hypothesized that selinexor would upregulate T cell checkpoint molecule expression, and thereby enhance the anti-tumor activity of antibodies targeting PD-1/PD-L1 or CTLA4. Human (A375, CHL-1) and murine (B16F10) melanoma cell lines expressed high levels of PD-L1 protein at baseline, and PD-L1 expression was induced following selinexor treatment in numerous other tumor cell lines (including HCT-116, MDA-MB-468, MV-4-11, OVCAR-8, and PC-3). Examination of lymphocytes revealed that selinexor also increased expression of PD-1 and CTLA4 by ∼2-fold. Mice bearing syngeneic B16F10 melanoma tumors treated with selinexor (15 mg/kg 2 x weekly) and anti-CTLA4 (250 μg, 2 x weekly) demonstrated a significant reduction in tumor growth rate (p = 0.0065) while monotherapy had no significant effect on tumor growth. Similar results were obtained in mice bearing B16F10 melanoma treated with the combination of selinexor + anti-PD-1 (200 μg, 2 x weekly, p < 0.034) or selinexor + anti-PD-L1 (100-200 μg, 2 x weekly, p < 0.001). Importantly, no weight loss or signs of toxicity were evident in any in vivo study. Further immunophenotypic analyses have been completed in animals receiving selinexor alone or in combination with anti-PD-L1. In combination treated mice, we observed a significantly increased percentage of splenic NK cells (p ≤ 0.050), and a significantly increased percentage of splenic Th1 T cells (p≤0.011), all compared to vehicle treated mice. Interestingly, combining selinexor with anti-PD-L1 significantly decreased the percentage of splenocytes that expressed PD-L1 (p<0.001). These changes are indicative of increased anti-tumor immune activity; however, they were accompanied by significantly increased percentages of myeloid cell subsets in combination treated mice (p ≤ 0.050). The immunologic significance of this myeloid cell expansion is currently under investigation. These data indicate that the efficacy of selinexor may be enhanced by disrupting immune checkpoints in effector cells (T and NK cells). This provides data in support of novel, evidenced-based combinations involving immunotherapy with XPO1 inhibition that deserve further investigation for advanced cancer. Citation Format: Matthew R. Farren, Reena Shakya, Rebecca Hennessey, Thomas Mace, Jennifer Yang, Omar Elnaggar, Gregory Young, Yosef Landesman, Robert Carlson, Sivan Elloul, Marsha Crochiere, Christin Burd, Gregory B. Lesinski. Selinexor, a selective inhibitor of nuclear export (SINE), enhances the in vivo efficacy of checkpoint blockade with antibodies targeting CTLA4 or PD-1/PD-L1 in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2319.

Abstract 2656: Combined inhibition of MEK and PI3K elicits anti-tumor activity in human cholangiocarcinoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Cholangiocarcinoma (CC) responds poorly to chemo- and immunotherapy and is nearly always fatal within one year. In recent years we have gained important insight into the signaling pathways that drive this cancer. These cancers are characterized by deregulated mitogen-activated protein kinase (MAPK), PI3 kinase/Akt and a dependence on the IL-6 axis of signal transduction. Importantly, we previously reported clinical responses for CC patients receiving single agent MEK inhibitors, indicating these agents have activity in this disease. We hypothesized that dual targeting of these pathways in CC using MEK162 and buparlisib would lead to potent antitumor and immunomodulatory activity, possibly circumventing resistance to single agent MEK162. In a panel of n = 7 human CC cell lines with diverse genetic profiles, constitutive phosphorylation of ERK (7/7) and Akt (2/7) was observed. Human CC cell lines displayed variable sensitivity to the growth inhibitory and pro-apoptotic effects of single agent MEK162 or buparlisib. CC cell lines with basal AKT phosphorylation (WITT, Mz-Cha-1) showed greater sensitivity to growth inhibition by buparlisib (IC50 10-20 μM), as compared to CC lines lacking pAKT (HuCCT1, HuH28, IC50 > 20μM). Immunoblot analysis confirmed decreased phosphorylated ERK (pERK) in the HuCCT1 and SNU-478 CC cell lines following treatment with MEK162. Culture supernatants from four separate human CC cell lines displayed significant reductions in IL-6, VEGF, and GM CSF in a concentration-dependent manner after treatment with MEK162. Immunomodulatory effects of MEK162 were also evident, independent of its tumor-intrinsic effects upon CC cell lines. Namely, it significantly reduced IL 6/GM-CSF driven MDSC differentiation (HLA-DRlo CD11b+ CD33+) from healthy normal donor PBMC in vitro. These observations were not due to cytotoxic activity as treatment with MEK162 for 72 hours did not reduce viability of bulk human PBMCs. Finally, to evaluate the effect of this treatment combination, in vivo studies were conducted in athymic mice bearing Mz-Cha1 or SNU-478 xenografts. Tumor bearing mice received daily oral administration of MEK162 (30 mg/kg), buparlisib (25 mg/kg), or both agents combined. Vehicle treated animals served as negative controls. Inhibition of tumor growth was observed following administration of single agent MEK162 or buparlisib as compared to control animals. This effect was further enhanced in the combination treated animals. Body weight of animals indicated this regimen was well-tolerated. Together, these data suggest that dual PI3K and MEK inhibition can target CC with varying genotypes and represents a promising therapeutic regimen with potential for direct antitumor activity and immunomodulation in CC. Citation Format: Jennifer Yang, Omar Elnaggar, Thomas Mace, Matthew Farren, Gregory Young, Patrice Lee, Kaitlin Keenan, Zheng Che, Jacob Kaufman, Denis Guttridge, David Carbone, Cynthia Timmers, Tanios Bekaii-Saab, Gregory Lesinski. Combined inhibition of MEK and PI3K elicits anti-tumor activity in human cholangiocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2656. doi:10.1158/1538-7445.AM2015-2656