Ashutosh Pal

Mechanism of Activation and Inhibition of the HER4/ErbB4 Kinase.

HER4/ErbB4 is a ubiquitously expressed member of the EGF/ErbB family of receptor tyrosine kinases that is essential for normal development of the heart, nervous system, and mammary gland. We report here crystal structures of the ErbB4 kinase domain in active and lapatinib-inhibited forms. Active ErbB4 kinase adopts an asymmetric dimer conformation essentially identical to that observed to be important for activation of the EGF receptor/ErbB1 kinase. Mutagenesis studies of intact ErbB4 in Ba/F3 cells confirm the importance of this asymmetric dimer for activation of intact ErbB4. Lapatinib binds to an inactive form of the ErbB4 kinase in a mode equivalent to its interaction with the EGF receptor. All ErbB4 residues contacted by lapatinib are conserved in the EGF receptor and HER2/ErbB2, which lapatinib also targets. These results demonstrate that key elements of kinase activation and inhibition are conserved among ErbB family members.

Molecular Imaging of EGFR Kinase Activity in Tumors with 124I-Labeled Small Molecular Tracer and Positron Emission Tomography

Positron emission tomography (PET) with epidermal growth factor receptor (EGFR) kinase-specific radiolabeled tracers could provide the means for noninvasive and repetitive imaging of heterogeneity of EGFR expression and signaling activity in tumors in individual patients before and during therapy with EGFR signaling inhibitors. We developed the synthesis and 124I-radiolabeling of the (E)-But-2-enedioic acid [4-(3-[124I]iodoanilino)-quinazolin-6-yl]-amide-(3-morpholin-4-yl-propyl)-amide (morpholino-[124I]-IPQA), which selectively, irreversibly, and covalently binds the adenosine-triphosphate-binding site to the activated (phosphorylated) EGFR kinase, but not to the inactive EGFR kinase. The latter was demonstrated using in silico modeling with crystal structures of the wild type and different gain-of-function mutants of EGFR kinases. Also, this was demonstrated by selective radiolabeling of the EGFR kinase domain with morpholino-[131I]-IPQA in A431 human epidermoid carcinoma cells and Western blot autoradiography. In vitro radiotracer accumulation and washout studies demonstrated a rapid accumulation and progressive retention postwashout of morpholino-[131I]-IPQA in A431 epidermoid carcinoma and in U87 human glioma cells genetically modified to express the EGFRvIII mutant receptor, but not in the wild-type U87MG glioma cells under serum-starved conditions. Using morpholino-[124I]-IPQA, we obtained noninvasive PET images of EGFR activity in A431 subcutaneous tumor xenografts, but not in subcutaneous tumor xenografts grown from K562 human chronic myeloid leukemia cells in immunocompromised rats and mice. Based on these observations, we suggest that PET imaging with morpholino-[124I]-IPQA should allow for identification of tumors with high EGFR kinase signaling activity, including brain tumors expressing EGFRvIII mutants and nonsmall-cell lung cancer expressing gain-of-function EGFR kinase mutants. Because of significant hepatobiliary clearance and intestinal reuptake of the morpholino-[124I]-IPQA, additional [124I]-IPQA derivatives with improved water solubility may be required to optimize the pharmacokinetics of this class of molecular imaging agents.

Activity of lapatinib is independent of EGFR expression level in HER2-overexpressing breast cancer cells.

Epidermal growth factor receptor (EGFR/ErbB1) and HER2 (ErbB2/neu), members of the ErbB receptor tyrosine kinase family, are frequently overexpressed in breast cancer and are known to drive tumor growth and progression, making them promising targets for cancer therapy. Lapatinib is a selective competitive inhibitor of both the HER2 and EGFR tyrosine kinases. Although lapatinib showed significant activity in patients with HER2-positive breast cancer, the role of EGFR in the response of breast cancer to lapatinib has not been defined. Here, we examined the role of EGFR expression levels in the sensitivity of HER2-overexpressing breast cancer cells to lapatinib. Depletion of EGFR by EGFR small-interfering RNA knockdown did not affect lapatinib sensitivity in these cells, whereas treated HER2 siRNA knockdown cells became more resistant to lapatinib. We conclude that the in vitro activity of lapatinib is not dependent on EGFR expression level in HER2-overexpressing breast cancer cells. [Mol Cancer Ther 2008;7(7):1846–50]

Detection of histone deacetylase inhibition by noninvasive magnetic resonance spectroscopy.

Histone deacetylase (HDAC) inhibitors are new and promising antineoplastic agents. Current methods for monitoring early response rely on invasive biopsies or indirect blood-derived markers. Our goal was to develop a magnetic resonance spectroscopy (MRS)–based method to detect HDAC inhibition. The fluorinated lysine derivative Boc-Lys-(Tfa)-OH (BLT) was investigated as a 19F MRS molecular marker of HDAC activity together with 31P MRS of endogenous metabolites. In silico modeling of the BLT-HDAC interaction and in vitro MRS studies of BLT cleavage by HDAC confirmed BLT as a HDAC substrate. BLT did not affect cell viability or HDAC activity in PC3 prostate cancer cells. PC3 cells were treated, in the presence of BLT, with the HDAC inhibitor p-fluoro-suberoylanilide hydroxamic acid (FSAHA) over the range of 0 to 10 μmol/L, and HDAC activity and MRS spectra were monitored. Following FSAHA treatment, HDAC activity dropped, reaching 53% of control at 10 μmol/L FSAHA. In parallel, a steady increase in intracellular BLT from 14 to 32 fmol/cell was observed. BLT levels negatively correlated with HDAC activity consistent with higher levels of uncleaved BLT in cells with inhibited HDAC. Phosphocholine, detected by 31P MRS, increased from 7 to 16 fmol/cell following treatment with FSAHA and also negatively correlated with HDAC activity. Increased phosphocholine is probably due to heat shock protein 90 inhibition as indicated by depletion of client proteins. In summary, 19F MRS of BLT, combined with 31P MRS, can be used to monitor HDAC activity in cells. In principle, this could be applied in vivo to noninvasively monitor HDAC activity. [Mol Cancer Ther 2006;5(5):1325–34]

Imaging Epigenetic Regulation by Histone Deacetylases in the Brain using PET/MRI with 18F-FAHA

Epigenetic modifications mediated by histone deacetylases (HDACs) play important roles in the mechanisms of different neurologic diseases and HDAC inhibitors (HDACIs) have shown promise in therapy. However, pharmacodynamic profiles of many HDACIs in the brain remain largely unknown due to the lack of validated methods for noninvasive imaging of HDAC expression-activity. In this study, dynamic PET/CT imaging was performed in 4 rhesus macaques using [(18)F]FAHA, a novel HDAC substrate, and [(18)F]fluoroacetate, the major radio-metabolite of [(18)F]FAHA, and fused with corresponding MR images of the brain. Quantification of [(18)F]FAHA accumulation in the brain was performed using a customized dual-tracer pharmacokinetic model. Immunohistochemical analyses of brain tissue revealed the heterogeneity of expression of individual HDACs in different brain structures and cell types and confirmed that PET/CT/MRI with [(18)F]FAHA reflects the level of expression-activity of HDAC class IIa enzymes. Furthermore, PET/CT/MRI with [(18)F]FAHA enabled non-invasive, quantitative assessment of pharmacodynamics of HDAC inhibitor SAHA in the brain.

Curcumin glucuronides: assessing the proliferative activity against human cell lines.

A gram scale synthesis of the glucuronide metabolites of curcumin were completed in four steps. The newly synthesized curcumin glucuronide compounds 2 and 3 along with curcumin 1 were tested and their anti-proliferative effects against KBM-5, Jurkat cell, U266, and A549 cell lines were reported. Biological data revealed that as much as 1 μM curcumin 1 exhibited anticancer activity and almost 100% cell kill was noted at 10 μM on two out of four cell lines; while curcumin mono-glucuronide 2 as well as di-glucuronide 3 displayed no suppression of cell proliferation.

Suberoylanilide hydroxamic acid blocks self-renewal and homotypic aggregation of inflammatory breast cancer spheroids

Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer (LABC). Patients with IBC commonly present with skin metastasis, which are observed microscopically as tumor emboli within dermal lymphatics. These metastatic tumor cells aberrantly overexpress E‐cadherin and exhibit the ability to undergo self‐renewal and are highly invasive. There are no therapeutics yet identified that target the structure and functions of IBC tumor emboli. The present studies evaluated the effects of the pan‐histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) using IBC tumor spheroids derived from established IBC cell lines and tumor spheroids derived from pleural effusion (PE) aspirates of patients with IBC and LABC, designated as PE‐IBC and PE‐LABC.

Monitoring Histone Deacetylase Inhibition In Vivo: Noninvasive Magnetic Resonance Spectroscopy Method

Histone deacetylase inhibitors (HDACis) are emerging as promising and selective antitumor agents. However, HDACis can lead to tumor stasis rather than shrinkage, in which case, traditional imaging methods are not adequate to monitor response. Consequently, novel approaches are needed. We have shown in cells that 19F magnetic resonance spectroscopy (MRS)-detectable levels of the HDAC substrate Boc-Lys-TFA-OH (BLT) are inversely correlated with HDAC activity. We extended our investigations to a tumor xenograft model. Following intraperitoneal injection of BLT, its accumulation within the tumor was monitored by in vivo 19F MRS. In animals treated with the HDACi suberoylanilide hydroxamic acid (SAHA), tumoral BLT levels were higher by 77% and 132% on days 2 and 7 of treatment compared with pretreatment levels (n = 6; p < .05). In contrast, tumoral BLT levels remained unchanged in control animals and in normal tissue. Thus, 19F MRS of BLT detected the effect of HDACi treatment as early as day 2 of treatment. Importantly, tumor size confirmed that SAHA treatment leads to inhibition of tumor growth. However, difference in tumor size reached significance only on day 6 of treatment. Thus, this work identifies BLT as a potential molecular imaging agent for the early noninvasive MRS detection of HDAC inhibition in vivo.

Detection of pancreatic carcinomas by imaging lactose-binding protein expression in peritumoral pancreas using [18F] fluoroethyl-deoxylactose PET/CT

Background Early diagnosis of pancreatic carcinoma with highly sensitive diagnostic imaging methods could save lives of many thousands of patients, because early detection increases resectability and survival rates. Current non-invasive diagnostic imaging techniques have inadequate resolution and sensitivity for detection of small size (∼2–3 mm) early pancreatic carcinoma lesions. Therefore, we have assessed the efficacy of positron emission tomography and computer tomography (PET/CT) imaging with β-O-D-galactopyranosyl-(1,4′)-2′-deoxy-2′-[18F]fluoroethyl-D-glucopyranose ([18F]FEDL) for detection of less than 3 mm orthotopic xenografts of L3.6pl pancreatic carcinomas in mice. [18F]FEDL is a novel radioligand of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), which is overexpressed in peritumoral pancreatic acinar cells. Methodology/Principal Findings Dynamic PET/CT imaging demonstrated rapid accumulation of [18F]FEDL in peritumoral pancreatic tissue (4.04±2.06%ID/g), bi-exponential blood clearance with half-lives of 1.65±0.50 min and 14.14±3.60 min, and rapid elimination from other organs and tissues, predominantly by renal clearance. Using model-independent graphical analysis of dynamic PET data, the average distribution volume ratio (DVR) for [18F]FEDL in peritumoral pancreatic tissue was estimated as 3.57±0.60 and 0.94±0.72 in sham-operated control pancreas. Comparative analysis of quantitative autoradiographic images and densitometry of immunohistochemically stained and co-registered adjacent tissue sections demonstrated a strong linear correlation between the magnitude of [18F]FEDL binding and HIP/PAP expression in corresponding regions (r = 0.88). The in situ analysis demonstrated that at least a 2–4 fold apparent lesion size amplification was achieved for submillimeter tumors and to nearly half a murine pancreas for tumors larger than 3 mm. Conclusion/Significance We have demonstrated the feasibility of detection of early pancreatic tumors by non-invasive imaging with [18F]FEDL PET/CT of tumor biomarker HIP/PAP over-expressed in peritumoral pancreatic tissue. Non-invasive non-invasive detection of early pancreatic carcinomas with [18F]FEDL PET/CT imaging should aid the guidance of biopsies and additional imaging procedures, facilitate the resectability and improve the overall prognosis.

Designing and Developing S100P Inhibitor 5-Methyl Cromolyn for Pancreatic Cancer Therapy

We have previously shown that the antiallergic drug cromolyn blocks S100P interaction with its receptor receptor for advanced glycation end product (RAGE) and improves gemcitabine effectiveness in pancreatic ductal adenocarcinoma (PDAC). However, the concentration required to achieve its effectiveness was high (100 μmol/L). In this study, we designed and synthesized analogs of cromolyn and analyzed their effectiveness compared with the parent molecule. An ELISA was used to confirm the binding of S100P with RAGE and to test the effectiveness of the different analogs. Analog 5-methyl cromolyn (C5OH) blocked S100P binding as well as the increases in NF-κB activity, cell growth, and apoptosis normally caused by S100P. In vivo C5OH systemic delivery reduced NF-κB activity to a greater extent than cromolyn and at 10 times lesser dose (50 mg vs. 5 mg). Treatment of mice-bearing syngeneic PDAC tumors showed that C5OH treatment reduced both tumor growth and metastasis. C5OH treatment of nude mice bearing orthotopic highly aggressive pancreatic Mpanc96 cells increased the overall animal survival. Therefore, the cromolyn analog, C5OH, was found to be more efficient and potent than cromolyn as a therapeutic for PDAC. Mol Cancer Ther; 12(5); 654–62. ©2013 AACR.