Sutapa Sinha

Pembrolizumab in patients with CLL and Richter transformation or with relapsed CLL

Chronic lymphocytic leukemia (CLL) patients progressed early on ibrutinib often develop Richter transformation (RT) with a short survival of about 4 months. Preclinical studies suggest that programmed death 1 (PD-1) pathway is critical to inhibit immune surveillance in CLL. This phase 2 study was designed to test the efficacy and safety of pembrolizumab, a humanized PD-1-blocking antibody, at a dose of 200 mg every 3 weeks in relapsed and transformed CLL. Twenty-five patients including 16 relapsed CLL and 9 RT (all proven diffuse large cell lymphoma) patients were enrolled, and 60% received prior ibrutinib. Objective responses were observed in 4 out of 9 RT patients (44%) and in 0 out of 16 CLL patients (0%). All responses were observed in RT patients who had progression after prior therapy with ibrutinib. After a median follow-up time of 11 months, the median overall survival in the RT cohort was 10.7 months, but was not reached in RT patients who progressed after prior ibrutinib. Treatment-related grade 3 or above adverse events were reported in 15 (60%) patients and were manageable. Analyses of pretreatment tumor specimens from available patients revealed increased expression of PD-ligand 1 (PD-L1) and a trend of increased expression in PD-1 in the tumor microenvironment in patients who had confirmed responses. Overall, pembrolizumab exhibited selective efficacy in CLL patients with RT. The results of this study are the first to demonstrate the benefit of PD-1 blockade in CLL patients with RT, and could change the landscape of therapy for RT patients if further validated. This trial was registered at www.clinicaltrials.gov as #NCT02332980.

Plumbagin inhibits tumorigenesis and angiogenesis of ovarian cancer cells in vivo

Angiogenesis is a hallmark of tumor development and metastatic progression, and anti‐angiogenic drugs targeting the VEGF pathway have shown to decrease the disease progression in cancer patients. In this study, we have analyzed the anti‐proliferative and anti‐angiogenic property of plumbagin in cisplatin sensitive, BRCA2 deficient, PEO‐1 and cisplatin resistant, BRCA2 proficient PEO‐4 ovarian cancer cells. Both PEO‐1 and PEO‐4 ovarian cancer cells are sensitive to plumbagin irrespective of BRCA2 status in both normoxia and hypoxia. Importantly, plumbagin treatment effectively inhibits VEGF‐A and Glut‐1 in PEO‐1 and PEO‐4 ovarian cancer cells. We have also analyzed the p53 mutant, cisplatin resistant, and BRCA2 proficient OVCAR‐5 cells. Plumbagin challenge also restricts the VEGF induced pro‐angiogenic signaling in HUVECs and subsequently endothelial cell proliferation. In addition, we observe a significant effect on tumor regression among OVCAR‐5 tumor‐bearing mice treated with plumbagin, which is associated with significant inhibition of Ki67 and vWF expressions. Plumbagin also significantly reduces CD31 expression in an ear angiogenesis assay. Collectively, our studies indicate that plumbagin, as an anti‐cancer agent disrupts growth of ovarian cancer cells through the inhibition of proliferation as well as angiogenesis.

Role of elongin-binding domain of von hippel lindau gene product on HuR-mediated VPF/VEGF mRNA stability in renal cell carcinoma

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a key mediator of angiogenesis for both physiological and pathological conditions. It is well established that the hypoxic induction of VPF/VEGF is in large part an increase in the stability of its mRNA. A Hu family ubiquitously expressed RNA-binding protein HuR has recently been shown to be important for VPF/VEGF mRNA stabilization. In renal cancer cells, the inactivation of the tumor suppressor protein von Hippel Lindau (VHL) leads to an increase in VPF/VEGF expression. VHL not only inhibits the transcription of VPF/VEGF but also plays a significant role in decreasing its mRNA stability. Here we delineate a possible mechanism by which VHL can control the function of HuR in order to regulate the stability of VPF/VEGF mRNA. The experiments presented here suggest that the association of the elongin-binding domain of VHL with a specific RNA-binding domain of HuR (RRM1) is important for the destabilizing function of VHL on VPF/VEGF mRNA.

Ephrin B2/EphB4 pathway in hepatic stellate cells stimulates Erk-dependent VEGF production and sinusoidal endothelial cell recruitment

Chemotaxis signals between hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) maintain hepatic vascular homeostasis and integrity and also regulate changes in sinusoidal structure in response to liver injury. Our prior studies have demonstrated that the bidirectional chemotactic signaling molecules EphrinB2 and EphB4 are expressed in HSC. The aim of our present study was to explore whether and how the EphrinB2/EphB4 system in HSC could promote SEC recruitment, which is essential for sinusoidal structure and remodeling. Stimulation of human HSC (hHSC) with chimeric agonists (2 microg/ml) of either EphrinB2 or EphB4 (EphrinB2 Fc or EphB4 Fc, respectively) significantly increased VEGF mRNA levels in hHSC as assessed by quantitative PCR, with respective small interfering RNAs for EphrinB2 and EphB4 inhibiting this increase (P < 0.05, n = 3). EphrinB2 agonist-induced increase in VEGF mRNA levels in hHSC was associated with increased phosphorylation of Erk and was significantly blocked by U0126 (20 microM), an inhibitor of MEK, which is a kinase upstream from Erk (P < 0.05, n = 3). The EphB4 agonist also significantly increased human VEGF promoter activity (P < 0.05, n = 3) as assessed by promoter reporter luciferase assay in transfected LX2-HSC. This was associated with upregulation of the vasculoprotective transcription factor, Kruppel-like factor 2 (KLF2). In Boyden chamber assays, conditioned media from hHSC stimulated with agonists of EphrinB2 or EphB4 increased SEC chemotaxis in a VEGF-dependent manner, compared with control groups that included basal media with agonists of EphrinB2, EphB4, or HSC-conditioned media from HSC in absence of agonist stimulation (P < 0.05, n = 3). EphB4 expression was detected in situ within liver sinusoidal vessels of rats after carbon tetrachloride-induced liver injury. In summary, activation of the EphrinB2/EphB4 signaling pathway in HSC promotes chemotaxis of SEC through a pathway that involves Erk, KLF2, and VEGF. These studies identify EphrinB2 or EphB4 as a key intermediary that links HSC signal transduction pathways with angiogenesis and sinusoidal remodeling.

Dopamine regulates phosphorylation of VEGF receptor 2 by engaging Src-homology-2-domain-containing protein tyrosine phosphatase 2

Vascular endothelial growth factor (VEGF)-induced receptor phosphorylation is the crucial step for initiating downstream signaling pathways that lead to angiogenesis or related pathophysiological outcomes. Our previous studies have shown that the neurotransmitter dopamine could inhibit VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR-2), endothelial cell proliferation, migration, microvascular permeability, and thus, angiogenesis. In this study, we address the mechanism by which VEGFR-2 phosphorylation is regulated by dopamine. Here, we demonstrate that D2 dopamine receptor (D2DR) colocalizes with VEGFR-2 at the cell surface. Dopamine pretreatment increases the translocation and colocalization of Src-homology-2-domain-containing protein tyrosine phosphatase (SHP-2) with D2DR at the cell surface. Dopamine administration leads to increased VEGF-induced phosphorylation of SHP-2 and this increased phosphorylation parallels the increased phosphatase activity of SHP-2. Active SHP-2 then dephosphorylates VEGFR-2 at Y951, Y996 and Y1059, but not Y1175. We also observe that SHP-2 knockdown impairs the dopamine-regulated inhibition of VEGF-induced phosphorylation of VEGFR-2 and, subsequently, Src phosphorylation and migration. Our data establish a novel role for SHP-2 phosphatase in the dopamine-mediated regulation of VEGFR-2 phosphorylation.

The neurotransmitter dopamine modulates vascular permeability in the endothelium

Background Vascular permeability factor/Vascular endothelial growth factor (VPF/VEGF), a multifunctional cytokine, is a potent inducer of vascular permeability, an important early step in angiogenesis. It is known that the neurotransmitter dopamine can inhibit VPF/VEGF mediated angiogenesis, in particular microvascular permeability, but the effectors of this action remain unclear. Results Here, we define the signaling pathway modulated by dopamine that inhibits VPF/VEGF induced vascular permeability in endothelial cells. Signals from VPF/VEGF lead to changes in the phosphorylation of tight junction protein zonula occludens (ZO-1) and adherens junction proteins like VE-cadherin and associated catenins, thus weakening endothelial cell-cell adhesion and increasing vascular permeability. We found VEGF receptor-2 (VEGFR-2) to be part of a multi-protein complex involving ZO-1, VE-cadherin and β-catenin. VPF/VEGF induced phosphorylations of VE-cadherin, β-catenin and ZO-1 were inhibited by dopamine treatment. Association of occludin with ZO-1 and ZO-1 with VE-cadherin were significantly inhibited by dopamine in VEGF treated cells. Furthermore, we identified Src as an important target for dopamine-mediated inhibition of VPF/VEGF induced permeability. Conclusion Taken together, our results provide molecular insights of dopamine function in the vascular endothelium and suggest a central role of Src in regulating key molecules that control vascular permeability.

Von Hippel-Lindau gene product modulates TIS11B expression in renal cell carcinoma: impact on vascular endothelial growth factor expression in hypoxia.

TIS11B belongs to a group of RNA-binding proteins (including TIS11/tristetraprolin and TIS11D) that share characteristic tandem CCCH-type zinc-finger domains and can be rapidly induced by multiple stimuli. TIS11B has been shown to regulate vascular endothelial growth factor (VEGF) mRNA stability in adrenocorticotropic hormone-stimulated primary adrenocortical cells. TIS11B has also been documented as a negative regulator of VEGF during development, but nothing has yet been reported in the context of human cancers. The Von Hippel-Lindau (VHL) tumor suppressor protein regulates VEGF gene expression at both the transcriptional and post-transcriptional levels in normoxia. However, whether it can do so in hypoxia is still unclear. Here, we report a unique regulatory function of VHL in VEGF expression in hypoxia that is mediated through modulation of TIS11B protein levels in renal cancer cells. In normoxia, we detected increased expression of the microRNA hsa-miR-29b in the VHL-overexpressing renal cancer cell line 786-O. We also show that this increased expression of hsa-miR-29b decreased TIS11B protein expression by post-transcriptional regulation in normoxia. In contrast, in hypoxia, increased TIS11B expression paralleled an increased TIS11B mRNA stability in VHL-overexpressing 786-O cells. This VHL-mediated TIS11B up-regulation in hypoxia may be important for TIS11B-regulated gene expression: we observed a down-regulation of VEGF mRNA in hypoxia in VHL-overexpressing cells compared with parental 786-O cells, and this effect was reversible by silencing TIS11B expression.

A Lipid-Modified Estrogen Derivative that Treats Breast Cancer Independent of Estrogen Receptor Expression through Simultaneous Induction of Autophagy and Apoptosis

It is a challenge to develop a universal single drug that can treat breast cancer at single- or multiple-stage complications, yet remains nontoxic to normal cells. The challenge is even greater when breast cancer–specific, estrogen-based drugs are being developed that cannot act against multistaged breast cancer complications owing to the cells differential estrogen receptor (ER) expression status and their possession of drug-resistant and metastatic phenotypes. We report here the development of a first cationic lipid-conjugated estrogenic derivative (ESC8) that kills breast cancer cells independent of their ER expression status. This ESC8 molecule apparently is nontoxic to normal breast epithelial cells, as well as to other noncancer cells. ESC8 induces apoptosis through an intrinsic pathway in ER-negative MDA-MB-231 cells. In addition, ESC8 treatment induces autophagy in these cells by interfering with the mTOR activity. This is the first example of an estrogen structure–based molecule that coinduces apoptosis and autophagy in breast cancer cells. Further in vivo study confirms the role of this molecule in tumor regression. Together, our results open new perspective of breast cancer chemotherapy through a single agent, which could provide the therapeutic benefit across all stages of breast cancer. Mol Cancer Res; 9(3); 364–74. ©2011 AACR.

The tumor suppressor axis p53/miR-34a regulates Axl expression in B-cell chronic lymphocytic leukemia: implications for therapy in p53-defective CLL patients.

The tumor suppressor axis p53/miR-34a regulates Axl expression in B-cell chronic lymphocytic leukemia: implications for therapy in p53-defective CLL patients

Structure-activity study to develop cationic lipid-conjugated haloperidol derivatives as a new class of anticancer therapeutics

Haloperidol (HP), a neuroleptic drug, shows high affinity toward σ receptors (SR). HP and reduced-HP at higher concentration were known to induce apoptosis in SR-overexpressing carcinomas and melanomas. Herein, we report the development of cationic lipid-conjugated haloperidol as a new class of anticancer therapeutics. In comparison to HP, the C-8 carbon chain analogue (HP-C8) showed significantly high, SR-assisted antiproliferative activity against cancer cells via caspase-3-mediated apoptosis and down-regulation of pAkt. Moreover, melanoma tumor aggressiveness in HP-C8-treated mice was significantly lower than that in HP-treated mice. HP-C8 simultaneously reduced Akt-protein level and increased Bax/Bcl-2 ratio in vascular endothelial cells, thereby indicating a possible protein kinase down-regulatory and apoptosis inducing role in tumor-associated vascular cells. In conclusion, we developed σ receptor-targeting cationic lipid-modified HP derivatives as a promising class of anticancer therapeutic that concurrently affects cancer and tumor environment associated angiogenic vascular cells through induction of apoptosis and Akt protein down-regulation.