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Cabozantinib; Differentiated thyroid cancer; Tyrosine kinase inhibitor Cabozantinib; Cáncer de tiroides diferenciado; Inhibidor de la tirosina quinasa Cabozantinib; Càncer de tiroides diferenciat; Inhibidor de la tirosina quinasa Background At an interim analysis (median follow-up, 6.2 months; n = 187), the phase 3 COSMIC-311 trial met the primary end point of progression-free survival (PFS): cabozantinib improved PFS versus a placebo (median, not reached vs. 1.9 months; p < .0001) in patients with previously treated radioiodine-refractory differentiated thyroid cancer (RAIR-DTC). The results from an exploratory analysis using an extended datacut are presented. Methods Patients 16 years old or older with RAIR-DTC who progressed on prior lenvatinib and/or sorafenib were randomized 2:1 to oral cabozantinib tablets (60 mg/day) or a placebo. Placebo patients could cross over to open-label cabozantinib upon radiographic disease progression. The objective response rate (ORR) in the first 100 randomized patients and the PFS in the intent-to-treat population, both according to Response Evaluation Criteria in Solid Tumors version 1.1 by blinded, independent review, were the primary end points. Results At the data cutoff (February 8, 2021), 258 patients had been randomized (cabozantinib, n = 170; placebo, n = 88); the median follow-up was 10.1 months. The median PFS was 11.0 months (96% confidence interval [CI], 7.4–13.8 months) for cabozantinib and 1.9 months (96% CI, 1.9–3.7 months) for the placebo (hazard ratio, 0.22; 96% CI, 0.15–0.32; p < .0001). The ORR was 11.0% (95% CI, 6.9%–16.9%) versus 0% (95% CI, 0.0%–4.1%) (p = .0003) with one complete response with cabozantinib. Forty placebo patients crossed over to open-label cabozantinib. Grade 3/4 treatment-emergent adverse events occurred in 62% and 28% of the cabozantinib- and placebo-treated patients, respectively; the most common were hypertension (12% vs. 2%), palmar–plantar erythrodysesthesia (10% vs. 0%), and fatigue (9% vs. 0%). There were no grade 5 treatment-related events. Conclusions At extended follow-up, cabozantinib maintained superior efficacy over a placebo in patients with previously treated RAIR-DTC with no new safety signals. This study was sponsored by Exelixis, Inc., (Alameda, California). We thank the patients and their families, the investigators, and the site staff. Writing and editorial assistance was provided by Alexus Rivas, PharmD, and Michael Raffin (Fishawack Communications, a part of Fishawack Health, Conshohocken, Pennsylvania) and was funded by Exelixis.

Introduction: Immune checkpoint inhibitors (ICI) have shown great promise in a wide spectrum of malignancies. However, responses are not always durable, and this mode of treatment is only effective in a subset of patients. As such, there exists an unmet need for novel approaches to bolster ICI efficacy.Areas covered: We review the role of the Tyro3, Axl, and Mer (TAM) receptor tyrosine kinases in promoting tumor-induced immune suppression and discuss the benefits that may be derived from combining ICI with TAM kinase-targeted tyrosine kinase inhibitors. We searched the MEDLINE Public Library of Medicine (PubMed) and EMBASE databases and referred to ClinicalTrials.gov for relevant ongoing studies.Expert opinion: Targeting of TAM kinases may improve the efficacy of immune checkpoint blockade. However, it remains to be determined whether this effect will be better achieved by the selective targeting of each TAM receptor, depending on the context, or by multi-receptor TAM inhibitors. Triple inhibition of all TAM receptors is more likely to be associated with an increased risk for adverse events. Clinical trial designs should use high-resolution clinical endpoints and proper control arms to determine the synergistic effects of combining TAM inhibition with immune checkpoint blockade.

Background Albumin-bilirubin (ALBI) grade is an objective measure of liver function for patients with hepatocellular carcinoma (HCC). The tyrosine kinase inhibitor cabozantinib is approved for patients with advanced HCC who have received prior sorafenib based on the phase 3 CELESTIAL trial (NCT01908426). Cabozantinib improved overall survival (OS) and progression-free survival (PFS) versus placebo in patients with previously treated HCC. Methods Patients were randomised 2:1 to receive cabozantinib 60 mg or placebo orally every day. Clinical outcomes in patients with ALBI grade 1 or 2 at baseline were evaluated in CELESTIAL. ALBI scores were retrospectively calculated based on baseline serum albumin and total bilirubin, with an ALBI grade of 1 defined as ≤ −2.60 score and a grade of 2 as a score of > −2.60 to ≤ −1.39. Results Cabozantinib improved OS and PFS versus placebo in both ALBI grade 1 (hazard ratio [HR] [95% CI]: 0.63 [0.46–0.86] and 0.42 [0.32–0.56]) and ALBI grade 2 (HR [95% CI]: 0.84 [0.66–1.06] and 0.46 [0.37–0.58]) subgroups. Adverse events were consistent with those in the overall population. Rates of grade 3/4 adverse events associated with hepatic decompensation were generally low and were more common among patients in the ALBI grade 2 subgroup. Discussion These results provide initial support of cabozantinib in patients with advanced HCC irrespective of ALBI grade 1 or 2. Trial registration number ClinicalTrials.gov number, NCT01908426.

Background: Patients with hepatocellular carcinoma (HCC) and Child-Pugh B liver cirrhosis have poor prognosis and are underrepresented in clinical trials. The CELESTIAL trial, in which cabozantinib improved overall survival (OS) and progression-free survival (PFS) versus placebo in patients with HCC and Child-Pugh A liver cirrhosis at baseline, was evaluated for outcomes in patients who had Child-Pugh B cirrhosis at Week 8. Methods: This was a retrospective analysis of adult patients with previously treated advanced HCC. Child-Pugh B status was assessed by the investigator. Patients were randomised 2:1 to cabozantinib (60 mg once daily) or placebo. Results: Fifty-one patients receiving cabozantinib and 22 receiving placebo had Child-Pugh B cirrhosis at Week 8. Safety and tolerability of cabozantinib for the Child-Pugh B subgroup were consistent with the overall population. For cabozantinib- versus placebo-treated patients, median OS from randomisation was 8.5 versus 3.8 months (HR 0.32, 95% CI 0.18–0.58), median PFS was 3.7 versus 1.9 months (HR 0.44, 95% CI 0.25–0.76), and best response was stable disease in 57% versus 23% of patients. Conclusions: These encouraging results with cabozantinib support the initiation of prospective studies in patients with advanced HCC and Child-Pugh B liver function.Clinical Trial Registration: NCT01908426

Albumin-bilirubin (ALBI) grade is an objective measure of liver function for patients with hepatocellular carcinoma (HCC). The tyrosine kinase inhibitor cabozantinib is approved for patients with advanced HCC who have received prior sorafenib based on the phase 3 CELESTIAL trial (NCT01908426). Cabozantinib improved overall survival (OS) and progression-free survival (PFS) versus placebo in patients with previously treated HCC.Patients were randomised 2:1 to receive cabozantinib 60 mg or placebo orally every day. Clinical outcomes in patients with ALBI grade 1 or 2 at baseline were evaluated in CELESTIAL. ALBI scores were retrospectively calculated based on baseline serum albumin and total bilirubin, with an ALBI grade of 1 defined as ≤ -2.60 score and a grade of 2 as a score of -2.60 to ≤ -1.39.Cabozantinib improved OS and PFS versus placebo in both ALBI grade 1 (hazard ratio [HR] [95% CI]: 0.63 [0.46-0.86] and 0.42 [0.32-0.56]) and ALBI grade 2 (HR [95% CI]: 0.84 [0.66-1.06] and 0.46 [0.37-0.58]) subgroups. Adverse events were consistent with those in the overall population. Rates of grade 3/4 adverse events associated with hepatic decompensation were generally low and were more common among patients in the ALBI grade 2 subgroup.These results provide initial support of cabozantinib in patients with advanced HCC irrespective of ALBI grade 1 or 2.ClinicalTrials.gov number, NCT01908426.

Photo-activatable carbon monoxide (CO)-releasing molecules (photoCORMs), have recently provided help to identify the salutary effects of CO in human pathophysiology. Among them notable is the ability of CO to sensitize chemotherapeutic-resistant cancer cells. Findings from our group have shown CO to mitigate drug resistance in certain cancer cells by the inhibition of cystathionine β-synthase (CBS), a key regulator of redox homeostasis in the cell. Diminution of the antioxidant capacity of cancer cells leads to sensitization to reactive oxygen species-producing drugs like doxorubicin and paclitaxel upon cotreatment with CO as well as in mitigating the drug effects of cisplatin. We hypothesize that the development of CO delivery techniques for coadministration with existing cancer treatment regimens may ultimately improve clinical outcomes in cancer therapy.

Antibodies conjugated to a photoactive transition metal carbonyl complex afford antigen-directed delivery of cytotoxic carbon monoxide to ovarian cancer cells., Carbon monoxide (CO)-releasing antibody conjugates were synthesized utilizing a photoactivatable CO-releasing molecule (photoCORM) and mouse monoclonal antibodies linked by a biotin-streptavidin system. Different monoclonal antibodies raised against different surface-expressed antigens that are implicated in ovarian cancer afforded a family of antibody-photoCORM conjugates (Ab-photoCORMs). In an immunosorbent/cell viability assay, Ab-photoCORMs accumulated onto ovarian cancer cells expressing the target antigens, delivering cytotoxic doses of CO in vitro. The results described here provide the first example of an “immunoCORM”, a proof-of-the-concept antibody-drug conjugate that delivers a gaseous molecule as a warhead to ovarian cancer.

Introduction: Ovarian cancer is one of the leading cause of morbidity and death among women, with a five-year relative survival rate of only 30% in patients diagnosed with distant metastasis. The ovarian cancer cells initially respond to first-line platinum drug cisplatin [cis-diamminedichloroplatinum(II) (CDDP)] treatment. But, they subsequently develop resistance to CDDP and eventually exhibit chemoresistance. Aldehyde dehydrogenase 1 family member A1 (ALDH1A1) is one of the key functional markers of ovarian cancer stem cells (CSCs) that confers cancer stemness and therapeutic resistance, and is associated with poor prognosis and patient survival. In this study, we have assessed the anticancer effects of the ALDH1A1 inhibitor, A37, in CDDP-resistant ovarian cancer cells in vitro. Experimental Methods: SK-OV-3-CDDP, cisplatin-resistant ovarian cancer cells were treated with different concentrations of the small molecule inhibitor of ALDH1A1, A37. We determined the cell proliferation using water-soluble tetrazolium salt (WST-1) assay at 24 and 48 h. The distribution of cell division phases by cell cycle analysis and oxygen consumption rate (OCR) via seahorse extracellular flux analysis were assessed by flow cytometry and seahorse XFe24 analyzer, respectively. Furthermore, we examined the protein expression of key signaling molecules by western blot analysis and cancer stemness by tumorsphere formation assay. Results: Treatment of SK-OV-3-CDDP cells with A37 significantly reduced the ovarian cancer cell proliferation. Interestingly, A37 induced cell cycle arrest as observed by an increase in G1 phase of the cell cycle. Additionally, A37 reduced the mitochondrial respiration of ovarian cancer cells as observed by the decrease in basal OCR. Moreover, A37 treatment markedly decreased the expression of WW domain containing transcription regulator 1 (WWTR1) protein [also called as transcriptional co-activator with PDZ-binding motif (TAZ)], which is a key downstream effector of mammalian Hippo signaling pathway that promotes cancer stemness, metastasis and chemoresistance. Importantly, A37 reduced the number and size of the tumorspheres. Conclusions: Our study suggests that inhibiting the ALDH1A1 activity using A37 reduced the cell proliferation and induced cell cycle arrest in CDPP-resistant ovarian cancer cells. The mechanism by which A37 elicits its anticancer effects on ovarian cancer cells include impairment in mitochondrial respiration that could alter cancer cell metabolism, and a decrease in WWTR1/TAZ expression and tumorsphere formation that could suppress cancer stemness. Our findings demonstrate that inhibition of ALDH1A1 could effectively eliminate the chemoresistant ovarian cancer cells, and therefore, new strategies targeting ALDH1A1 could lead to the development of novel therapeutics for aggressive chemoresistant ovarian cancer.

Ovarian cancer is the leading cause of death among all gynecological malignancies due to the development of acquired chemoresistance and disease relapse. Although the role of cancer stem cells (CSCs), a subset of tumor cells with the self-renewal and differentiation capabilities, in therapeutic resistance is beginning to be better understood, the significance of epigenetic regulatory mechanisms responsible for integrating the stemness with drug resistance remain poorly understood. Here we identified that lysine demethylase KDM3A as a critical regulator of ovarian cancer stemness and cisplatin resistance by inducing the expressions of pluripotent molecules Sox2 and Nanog and anti-apoptotic B-cell lymphoma 2 (Bcl-2), respectively. In addition, KDM3A induces ovarian cancer growth while antagonizing cellular senescence by repressing the expression of cyclin-dependent kinase inhibitor, p21Waf1/Cip1. The underlying mechanism of the noted biological processes include KDM3A-mediated stimulation of Sox2 expression, and demethylating p53 protein and consequently, modulating its target genes such as Bcl-2 and p21Waf1/Cip1 expression. Consistently, KDM3A depletion inhibited the growth of subcutaneously implanted cisplatin-resistant human ovarian cancer cells in athymic nude mice. Moreover, KDM3A is abundantly expressed and positively correlated with Sox2 expression in human ovarian cancer tissues. In brief, our findings reveal a novel mechanism by which KDM3A promotes ovarian CSCs, proliferation and chemoresistance and thus, highlights the significance of KDM3A as a novel therapeutic target for resistant ovarian cancer.

// Flavio Curnis 1,* , Alice Dallatomasina 1,* , Mimma Bianco 1,* , Anna Gasparri 1 , Angelina Sacchi 1 , Barbara Colombo 1 , Martina Fiocchi 1 , Laura Perani 2 , Massimo Venturini 2 , Carlo Tacchetti 2 , Suvajit Sen 3 , Ricardo Borges 4 , Eleonora Dondossola 1 , Antonio Esposito 2,5 , Sushil K. Mahata 6 and Angelo Corti 1,5 1 Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy 2 Experimental Imaging Center, IRCCS San Raffaele Scientific Institute, Milan, Italy 3 University of California, Los Angeles, CA, USA 4 La Laguna University, Tenerife, Spain 5 Vita-Salute San Raffaele University, Milan, Italy 6 VA San Diego Healthcare System and University of California, San Diego, La Jolla, CA, USA * These authors have contributed equally to this work Correspondence to: Flavio Curnis, email: // Angelo Corti, email: // Keywords : chromogranin A, angiogenesis, tumor perfusion, endothelial cells, protease-nexin-1 Received : September 9, 2016 Accepted : September 17, 2016 Published : September 24, 2016 Abstract Chromogranin A (CgA), a neuroendocrine secretory protein, and its fragments are present in variable amounts in the blood of normal subjects and cancer patients. We investigated whether circulating CgA has a regulatory function in tumor biology and progression. Systemic administration of full-length CgA, but not of fragments lacking the C-terminal region, could reduce tumor growth in murine models of fibrosarcoma, mammary adenocarcinoma, Lewis lung carcinoma, and primary and metastatic melanoma, with U-shaped dose-response curves. Tumor growth inhibition was associated with reduction of microvessel density and blood flow in neoplastic tissues. Neutralization of endogenous CgA with antibodies against its C-terminal region (residues 410-439) promoted tumor growth. Structure-function studies showed that the C-terminal region of CgA contains a bioactive site and that cleavage of this region causes a marked loss of anti-angiogenic and anti-tumor potency. Mechanistic studies showed that full-length CgA could induce, with a U-shaped dose-response curve, the production of protease nexin-1 in endothelial cells, a serine protease inhibitor endowed of anti-angiogenic activity. Gene silencing or neutralization of protease nexin-1 with specific antibodies abolished both anti-angiogenic and anti-tumor effects of CgA. These results suggest that circulating full-length CgA is an important inhibitor of angiogenesis and tumor growth, and that cleavage of its C-terminal region markedly reduces its activity. Pathophysiological changes in CgA blood levels and/or its fragmentation might regulate disease progression in cancer patients.

Cisplatin resistance remains a major impediment to effective treatment of ovarian cancer. Despite initial platinum responsiveness, thiol-containing peptides and proteins, glutathione (GSH) and metallothionein (MT), bind and inactivate cisplatin in cancer cells. Indeed, high levels of GSH and MT in ovarian cancers impart cisplatin resistance and are predictive of poor prognosis. Cystathionine β-synthase (CBS), an enzyme involved in sulfur metabolism, is overexpressed in ovarian cancer tissues and is itself associated with cisplatin resistance. Treatment with exogenous carbon monoxide (CO), a known inhibitor of CBS, may mitigate cisplatin resistance in ovarian cancer cells by attenuation of GSH and MT levels. Using a photo-activated CO-releasing molecule (photoCORM), [Mn(CO)3(phen)(PTA)]CF3SO3 (phen = 1,10-phenanthroline, PTA = 1,3,5-triza-7-phosphaadamantane) we assessed the ability of CO to sensitize established cisplatin-resistant ovarian cancer cell lines to cisplatin. Cisplatin-resistant cells, treated with both cisplatin and CO, exhibited significantly lower cell viability and increased poly (ADP-ribose) polymerase (PARP) cleavage versus those treated with cisplatin alone. These cisplatin-resistant cell lines overexpressed CBS and had increased steady state levels of GSH and expression of nuclear MT. Both CO treatment and lentiviral-mediated silencing of CBS attenuated GSH and nuclear MT expression in cisplatin resistant cells. We have demonstrated that CO, delivered from a photoCORM, sensitizes established cisplatin-resistant cell lines to cisplatin. Furthermore, we have presented strong evidence that the effects of CO in circumventing chemotherapeutic drug resistance is at least in part mediated by the inactivation of endogenous CBS.

Cystathionine β-synthase (CBS) is an enzyme in the transulfuration pathway that can catalyze the condensation of homocysteine (Hcy) and cysteine (Cys) to hydrogen sulfide (H2S) and cystathionine (CTH). CBS-derived H2S is important in angiogenesis and drug resistance in colon and ovarian cancers, respectively. However, the mechanisms by which cancer cell-derived H2S is utilized by cancer cells as a protective agent against host-derived activated macrophages are not yet investigated. This study investigated the mechanistic role of CBS-derived H2S in the protection of human breast cancer (HBC) cells against activated macrophages. HBC patient-derived tissue arrays and immunoblot analysis of HBC cells exhibited significantly increased levels of CBS when compared with their normal counterparts. This was associated with increased levels of H2S and CTH. Silencing of CBS in HBC cells caused a significant decrease in the levels of H2S and CTH but did not affect the growth of these cells per se, in in vitro cultures. However CBS-silenced cells exhibited significantly reduced growth in the presence of activated macrophages and in xenograft models. This was associated with an increase in the steady state levels of reactive aldehyde-derived protein adducts. Exogenous addition of H2S countered the effects of CBS silencing in the presence of macrophages. Conversely overexpression of CBS in human breast epithelial (HBE) cells (which do not naturally express CBS) protected them from activated macrophages, which were otherwise susceptible to the latter.

Drug resistance is a major impediment to effective treatment of breast cancer. Compared to normal cells, cancer cells have an increased antioxidant potential due to an increased ratio of reduced to oxidized glutathione (GSH/GSSG). This is known to confer therapeutic resistance. Here, we have identified a mechanism, unique to breast cancer cells, whereby cystathionine β-synthase (CBS) promotes elevated GSH/GSSG. Lentiviral silencing of CBS in human breast cancer cells attenuated GSH/GSSG, total GSH, nuclear factor erythroid 2-related factor 2 (Nrf2), and processes downstream of Nrf2 that promote GSH synthesis and regeneration of GSH from GSSG. Carbon monoxide (CO) reduced GSH/GSSG in three breast cancer cell lines by inhibiting CBS. Furthermore, CO sensitized breast cancer cells to doxorubicin. These results provide insight into mechanism(s) by which CBS increases the antioxidant potential and the ability for CO to inhibit CBS activity to alter redox homeostasis in breast cancer, increasing sensitivity to a chemotherapeutic.

Since the discovery in 1987 that vascular endothelial cells are able to synthesize nitric oxide (NO) from l -arginine, the existence of this biochemical pathway in many other cell types has been thoroughly documented, and its relevance in biology has become apparent. NO, a free-radical gas, synthesized by a family of isoenzymes called NO synthases, was shown to play a key role as a cell-signaling molecule in the vascular nervous and immune systems. It also exhibited a cytostatic/cytotoxic property at higher concentrations as generated by activated macrophages and endothelial cells. The role of NO in cancer biology and more so in breast cancer, however, started to be elucidated only in 1995 by Thomsen et al. (1995). In this chapter, we will review some of the key aspects of endogenous NO as produced in breast cancer cells and breast tumors. We will allude to the early studies that described NO production in breast tumor tissues and breast cancer cells and point to some of its proposed functions in breast cancer homeostasis. We will discuss how these findings could be of therapeutic benefit and finally end with some information on how a mechanistic cross talk between endogenously produced NO and another gaseous signaling molecule, hydrogen sulfide (H2S), in breast cancer cells may open up new opportunities for intervention. We will also discuss one potential mechanism of infiltration of tumor-associated macrophages in breast cancer and its clinical significance.

Background ALBI grade is an objective measure of liver function for patients with HCC; higher ALBI grade is associated with worse prognosis. In the phase 3 CELESTIAL trial (NCT01908426), cabozantinib, an inhibitor of MET, VEGFR, and AXL, significantly improved overall survival (OS) and progression-free survival (PFS) vs placebo in patients with previously treated HCC and is now approved for patients with HCC who received prior sorafenib. Here, we evaluate outcomes based on ALBI grade in the CELESTIAL trial. Methods 707 patients were randomized 2:1 to cabozantinib (60 mg daily) or placebo. Eligible patients had HCC, Child-Pugh score A, and ECOG PS ≤ 1. Patients received prior sorafenib and ≤2 lines of prior systemic therapy for HCC. Baseline ALBI score was calculated from serum albumin and total bilirubin measured centrally, and was used to determine ALBI grade (Johnson, J Clin Oncol. 2015;33:550-8). Results At baseline, 186 patients (40%) were ALBI grade 1 and 282 (60%) were ALBI grade 2 in the cabozantinib arm; 182 patients (43%) were ALBI grade 1 and 133 (56%) were ALBI grade 2 in the placebo arm. Two patients in each arm were ALBI grade 3. Patients with ALBI grade 1 had better ECOG PS (61% ECOG 0 & 39% ECOG 1) vs those with ALBI grade 2 (48% ECOG 0 & 52% ECOG 1). In patients with ALBI grade 1, median OS was 17.5 months with cabozantinib vs 11.4 months with placebo (HR 0.63, 95% CI 0.46-0.86). In patients with ALBI grade 2, median OS was 8.0 months with cabozantinib vs 6.4 months with placebo (HR 0.84, 95% CI 0.66-1.06). In patients with ALBI grade 1, median PFS was 6.5 months with cabozantinib vs 1.9 months with placebo (HR 0.42, 95% CI 0.32-0.56) and in patients with ALBI grade 2, median PFS was 3.7 months with cabozantinib vs 1.9 months with placebo (HR 0.46, 95% CI 0.37-0.58). Common grade 3/4 adverse events in both groups were consistent with the overall population. Treatment related discontinuations in the cabozantinib arm were 12% and 19% for patients with ALBI grade 1 and 2. Conclusions Patients treated with cabozantinib had longer PFS and OS vs patients receiving placebo, regardless of ALBI grade. Outcomes were generally better in patients with ALBI grade 1 vs 2. Clinical trial identification NCT01908426 (Other Study ID Numbers: XL184-309). Editorial acknowledgement Suvajit Sen (Exelixis). Legal entity responsible for the study Exelixis. Funding Exelixis. Disclosure S.L. Chan: Advisory / Consultancy: Amgen. R. Miksad: Full / Part-time employment: Flatiron Health; Shareholder / Stockholder / Stock options: Flatiron Health; Research grant / Funding (institution): Exelixis, Bayer. I. Cicin: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: Eli Lilly; Advisory / Consultancy: Merck Sharp & Dohme Corp; Advisory / Consultancy: F. Hoffmann-La Roche; Advisory / Consultancy: Pfizer. H.J. Klumpen: Advisory / Consultancy: IPSEN; Research grant / Funding (institution), Local PI for clinical trial from the following organizations: IPSEN Exelexis, ITM, SIRTEX, Taiho, BAYER, DAICHII, Novartis, BTG. S. Kim: Research grant / Funding (institution): Genentech, Roche, Pfizer; Advisory / Consultancy: Amgen, Bayer, Boehringer Ingelheim, MSD, Novartis, Roche, Sanofi, Servier. J. Youkstetter: Shareholder / Stockholder / Stock options, Full / Part-time employment: Exelixis. S. Sen: Full / Part-time employment, Self and spouse: Exelixis; Shareholder / Stockholder / Stock options, Self and spouse: Exelixis. A-L. Cheng: Advisory / Consultancy: Bayer Schering Pharma; Advisory / Consultancy: Bristol-Myers Squibb; Honoraria (self), Advisory / Consultancy: Eisai; Honoraria (self), Advisory / Consultancy: Merck Serono; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (self), Advisory / Consultancy: Ono Pharmaceutica; Advisory / Consultancy: Exelixis; Advisory / Consultancy: Nucleix; Honoraria (self), Advisory / Consultancy: Roche/Genentech; Honoraria (self), Advisory / Consultancy: IQVIA; Honoraria (self): Merck Sharp Dohme. A.B. El-Khoueiry: Research grant / Funding (self): AstraZeneca; Research grant / Funding (self): Astex; Advisory / Consultancy: Bayer; Advisory / Consultancy: BMS; Advisory / Consultancy: Agenus; Advisory / Consultancy: Merck; Advisory / Consultancy: EISAI; Advisory / Consultancy: Pieris; Advisory / Consultancy: EMD Serono; Advisory / Consultancy: Exelixis; Advisory / Consultancy: Roche/Genentech; Advisory / Consultancy: Apeiron. T. Meyer: Advisory / Consultancy: BMS, BAYER, EISAI, BTG, AZ, BEIGENE, TARVEDA, MSD; Research grant / Funding (institution): BTG BAYER. R.K. Kelley: Advisory / Consultancy: Agios, AstraZeneca, Bayer, BMS (funding to institution) IDMC: Genentech/Roche (funding to self); Research grant / Funding (institution): Agios, AstraZeneca, Bayer, BMS, Eli Lilly, Exelixis, EMD Serono, Medimmune, Merck, Novartis, QED, Taiho. G.K. Abou-Alfa: Research grant / Funding (self): ActaBiologica,; Advisory / Consultancy, Research grant / Funding (self): Agios,; Research grant / Funding (self): Array,; Advisory / Consultancy, Research grant / Funding (self): AstraZeneca; Advisory / Consultancy, Research grant / Funding (self): Bayer,; Advisory / Consultancy, Research grant / Funding (self): Beigene,; Advisory / Consultancy, Research grant / Funding (self): BMS,; Research grant / Funding (self): Casi,; Research grant / Funding (self): Celgene,; Research grant / Funding (self): Exelixis,; Research grant / Funding (self): Genentech,; Research grant / Funding (self): Halozyme,; Research grant / Funding (self): Incyte,; Research grant / Funding (self): Lilly,; Research grant / Funding (self): Mabvax,; Research grant / Funding (self): Novartis,; Research grant / Funding (self): OncoQuest,; Research grant / Funding (institution): Polaris Puma; Research grant / Funding (self): QED,; Research grant / Funding (self): Roche; Advisory / Consultancy: 3DMedcare,Agios, Alignmed, Amgen, Antengene, Aptus, Aslan, Astellas, AstraZeneca, Bayer, Beigene, Bioline, BMS, Boston Scientifc, Bridgebio, Carsgen, Celgene, Casi, Cipla, CytomX, Daiichi, Debio, Delcath, Eisai, Exelixis, Flatiron, Genoscience, Halozyme. All other authors have declared no conflicts of interest.

423 Background: AFP response, defined as a decrease in serum levels of the tumor marker AFP after therapy, may be associated with improved survival of patients (pts) with HCC treated with locoregional or systemic therapy, and high baseline AFP levels may be associated with poor prognosis. In the phase III CELESTIAL trial (NCT01908426), C, an inhibitor of MET, VEGFR, and AXL, significantly improved overall survival (OS) and progression-free survival (PFS) versus P in pts with previously treated advanced HCC. Here we evaluate clinical outcomes with C in CELESTIAL based on AFP response or progression on treatment. Methods: 707 pts were randomized 2:1 to receive C (60 mg daily) or P. Eligible patients had a pathologic diagnosis of HCC, Child-Pugh score A, and ECOG PS ≤ 1. Pts received prior sorafenib and ≤ 2 lines of prior systemic therapy for HCC. Serum AFP levels were measured centrally at baseline and every 8 weeks thereafter. Outcomes were evaluated for pts with baseline AFP ≥ 20 ng/mL based on AFP response ( ≥ 20% decrease from baseline) or progression ( ≥ 20% increase from baseline) at Week 8. This definition of AFP response has been used in previous studies but requires further validation in large prospective studies. Results: Overall, 331 pts (70%) in the C arm and 160 (68%) in the P arm had baseline AFP ≥ 20 ng/mL; among these pts, 236 (71%) and 111 (69%), respectively, were evaluable for AFP response at week 8. Among evaluable pts, 117 pts (50%) in the C arm vs 14 (13%) in the P arm had an AFP response, and 72 (31%) vs 75 (68%) had AFP progression. Median OS with C was 16.1 mo for pts with an AFP response versus 9.1 mo for pts without a response (HR 0.61, 95% CI 0.45-0.84), and median PFS with C was 7.3 mo versus 4.0 mo (HR 0.55, 95% CI 0.41-0.74). For pts with AFP progression, median OS with C was 8.1 mo, and median PFS with C was 3.6 mo. Hazard ratios for OS and PFS with C also favored AFP responders over non-responders when analyzed using best response through week 24. Conclusions: The AFP response rate was higher with C versus P, and AFP response was associated with longer OS and PFS with C for pts with previously treated advanced HCC. On-treatment AFP changes warrant further evaluation as a biomarker of response. Clinical trial information: NCT01908426.

It is established that high concentrations of nitric oxide(1) (NO), as released from activated macrophages, induce apoptosis in breast cancer cells. In this study, we assessed the potential of a light-activated NO donor [(Me2bpb)Ru(NO)(Resf)], a recently reported apoptototic agent, in suppressing the anchorage independent growth potentials of an aggressive human breast cancer cell line. Our results demonstrated the down regulation of anchorage independent growth by light activated NO treatment in the aggressive human breast cancer cell line MDA-MB-231 and afforded insight into the associated mechanism(s). The investigation revealed an up-regulation of the bioactivity of catalase with an accompanied reduction in the endogenous levels of H2O2, a direct substrate of catalase and a recently identified endogenous growth modulator in breast cancer cells. An earlier publication reported that endogenous superoxide (O2(-)) in human breast cancer cells constitutively inhibits catalase bioactivity (at the level of its protein), resulting in increased H2O2 levels. Interestingly in this study, O2(-) was also found to be down- regulated following NO treatment providing a basis for the observed increase in catalase bioactivity. Cells silenced for the catalase gene exhibited compromised reduction in anchorage independent growth upon light activated NO treatment. Collectively this study detailed a mechanistic cross talk between exogenous NO and endogenous ROS in attenuating anchorage independent growth.

Endothelial cells in the utero-placental circulation play an important physiological role in maintaining the fetoplacental vessels in a vasodilated state as these vessels are non-innervated. These endothelial cells produce both prostacyclin and nitric oxide which in addition to causing vasodilation also prevent platelet aggregation and adhesion of platelets to endothelial cells. Most investigators are of the opinion that energy metabolism of endothelial cells and ATP generation is mainly glycolytic. Glycolytic activity in endothelial cells is increased during proliferation to maintain ATP at normal levels by an increase in the expression of the glucose transporter. More recent studies have reported the existence of a functional F1F0 ATP synthase on the surface of HUVEC and it has been found to be enzymatically active in the synthesis of ATP. Additional studies utilizing very early passage HUVEC need to be carried out to ascertain the relative contribution of oxidative phosphorylation compared with the glycolytic pathway for ATP synthesis in normal pregnancy as well as in abnormal states like preeclampsia, diabetes, intrauterine injection as well as intrauterine growth restriction.

Accumulating evidence suggests that abnormal levels of homocysteine are associated with vascular dysfunctions, cancer cell proliferation and various neurodegenerative diseases. With respect to the latter, a perturbation of transition metal homeostasis and an inhibition of catalase bioactivity have been reported. Herein, we report on some of the molecular bases for the cellular toxicity of homocysteine and demonstrate that it induces the formation of sulfcatalase, an irreversible inactive state of the enzyme, without the intervention of hydrogen sulfide. Initially, homocysteine reacts with native catalase and/or redox-active transition metal ions to generate thiyl radicals that mediate compound II formation, a temporarily inactive state of the enzyme. Then, the ferryl centre of compound II intervenes into the unprecedented S-oxygenation of homocysteine to engender the corresponding sulfenic acid species that further participates into the prosthetic heme modification through the formation of an unusual Fe(II) sulfonium. In addition, our ex cellulo studies performed on cancer cells, models of neurodegenerative diseases and ulcerative colitis suggest the likelihood of this scenario in a subset of cancer cells, as well as in a cellular model of Parkinson's disease. Our findings expand the repertoire of heme modifications promoted by biological compounds and point out another deleterious trait of disturbed homocysteine levels that could participate in the aetiology of these diseases., High levels of homocysteine in cells are linked to pathological states. Here, the authors report that homocysteine inactivates catalase by modifying the heme group, impairing cellular redox homeostasis, and show that this modification occurs in cancer cells and in a cellular model of Parkinson's disease.

Context: Biochemical weakening of the amnion is a major factor preceding preterm premature rupture of membranes (PPROMs), leading to preterm birth. Activation of matrix metalloproteinases (MMPs) is known to play a key role in collagen degradation of the amnion; however, epithelial to mesenchymal transition (EMT) that is also induced by MMP activation has not been investigated as a mechanism for amnion weakening. Objective: To measure amniotic EMT associated with vaginal delivery (VD) compared with unlabored cesarean sections (CSs), and to assess changes in amniotic mechanical strength with pharmacologic inhibitors and inducers of EMT, thus testing the hypothesis that EMT is a key biochemical event that promotes amniotic rupture. Findings: (1) Amnions taken from VD contained a significantly increased number of mesenchymal cells relative to epithelial cells compared with unlabored CS by fluorescence-activated cell sorting analysis (60% vs 10%); (2) tumor necrosis factor (TNF)–α stimulation of amniotic epithelial cells increased expression of the mesenchymal marker vimentin after 2 days; (3) EMT inhibitor, etodolac, significantly increased the time and mechanical pressure required to rupture the amnion; and (4) TNF-α and another pharmacologic EMT inducer, ethacridine, decreased the time and mechanical pressure required for amnion rupture, further confirming that the mesenchymal phenotype significantly weakens the amnion. Conclusions: This work demonstrated amniotic cell EMT was associated with labor and EMT decreased the tensile strength of the amnion. These findings suggest a role for EMT in the pathophysiology of PPROM and may provide a basis for development of therapies to prevent preterm labor., We evaluated EMT in the human amnion and found that labor and PPROM are associated with increased amniotic EMT compared with unlabored cesarean delivery.

We assessed the catalase bioactivity and hydrogen peroxide (H₂O₂) production rate in human breast cancer (HBC) cell lines and compared these with normal human breast epithelial (HBE) cells. We observed that the bioactivity of catalase was decreased in HBC cells when compared with HBE cells. This was also accompanied by an increase in H₂O₂ steady-state levels in HBC cells. Silencing the catalase gene led to a further increase in the steady-state level of H₂O₂ which was also accompanied by an increase in growth rate of HBC cells. Catalase activity was up regulated on treatment with superoxide (O₂⁻) scavengers such as pegylated SOD (PEG-SOD, indicating inhibition of catalase by the increased O₂⁻ produced by HBC cells. Transfection of either catalase or glutathione peroxidase to HBC cells decreased intracellular H₂O₂ levels and led to apoptosis of these cells. The H₂O₂ produced by HBC cells inhibited PP2A activity accompanied by increased phosphorylation of Akt and ERK1/2. The importance of catalase bioactivity in breast cancer was further confirmed as its bioactivity was also decreased in human breast cancer tissues when compared to normal breast tissues. We conclude that inhibition of catalase bioactivity by O₂⁻ leads to an increase in steady-state levels of H₂O₂ in HBC cells, which in turn inhibits PP2A activity, leading to phosphorylation of ERK 1/2 and Akt and resulting in HBC cell proliferation.

Objective— Tumor necrosis factor-α (TNFα) may change from a stimulator of reversible activation of endothelial cells (ECs) to a killer when combined with cycloheximide (CHX). The means by which endothelial cells are destined to either the survival pathway or the apoptotic pathway are not fully understood. We investigated the role of p38 mitogen-activated protein kinase (MAPK) and protein phosphatase 2A (PP2A) activation and their regulation of 4E-BP1 stability in ECs to determine whether this pathway contributes to apoptosis induced by TNFα and CHX. Methods and Results— Apoptosis was induced in human umbilical vein ECs (HUVECs) by treating them with a combination of TNFα and CHX (TNFα/CHX). Activation of p38 MAPK was increased in HUVECs undergoing apoptosis, which was associated with degradation of eukaryotic initiation factor 4A regulator 4E-BP1 in a p38 MAPK–dependent manner. CHX attenuated a TNFα-stimulated increase in the expression and activity of PP2A. Silencing PP2A expression with small interfering RNA transfection mimicked CHX sensitization, increasing HUVEC apoptosis with TNFα stimulation and suggesting a protective role for PP2A in the apoptotic process. Conclusion— Our data suggest that (1) TNFα stimulates PP2A and HUVECs elude apoptosis by PP2A-dependent dephosphorylation of p38 MAPK, and (2) CHX-induced inhibition of PP2A leads to maintenance of p38 activity and degradation of 4E-BP1, resulting in enhanced TNFα-induced apoptosis.

Cystathionine beta-synthase catalyzes the condensation of serine and homocysteine to yield cystathionine and is the single most common locus of mutations associated with homocystinuria. In this study, we have examined the kinetic consequences of a pair of linked patient mutations, P78R/K102N, that are housed in the catalytic core of the protein and compared it to the effects of the corresponding single mutations. The P78R mutation affords purification of a mixture of higher order oligomers, P78R-I, which resembles the mixed quaternary state associated with wild-type enzyme. However, unlike wild-type enzyme, P78R-I converts over time to P78R-II, which exists predominantly as a full-length dimer. The specific activities of the K102N, P78R-I, and P78R-II mutants in the absence of AdoMet are approximately 3-, 9-, and 3-fold lower than of wild-type enzyme and are stimulated 2.9-, 2.5-, and 1.4-fold respectively by AdoMet. However, when linked, the specific activity of the resulting double mutant is comparable to that of wild-type enzyme but it is unresponsive to AdoMet, revealing that interactions between the two sites modulate the phenotype of the enzyme. Steady-state kinetic analysis for the double mutant reveals a sigmoidal dependence on homocysteine that is not observed with wild-type enzyme, which is ascribed to the mutation at the K102 locus and indicates changes in subunit interactions. Hydrogen-deuterium mass spectrometric analysis reveals that, even in the absence of AdoMet, the double mutant is locked in an activated conformation that is observed for wild-type enzyme in the presence of AdoMet, providing a structural rationale for loss of this allosteric regulation. To our knowledge, this is the first example of mutations in the catalytic core of cystathionine beta-synthase that result in failure of AdoMet-dependent regulation. Furthermore, analysis of individual single mutations has permitted, for the first time, partial kinetic characterization of a full-length dimeric form of human cystathionine beta-synthase.

Aggressive cancers exhibit an efficient conversion of high amounts of glucose to lactate accompanied by acid secretion, a phenomenon popularly known as the Warburg effect. The acidic microenvironment and the alkaline cytosol create a proton-gradient (acid gradient) across the plasma membrane that represents proton-motive energy. Increasing experimental data from physiological relevant models suggest that acid gradient stimulates tumor proliferation, and can also support its energy needs. However, direct biochemical evidence linking extracellular acid gradient to generation of intracellular ATP are missing. In this work, we demonstrate that cancer cells can synthesize significant amounts of phosphate-bonds from phosphate in response to acid gradient across plasma membrane. The noted phenomenon exists in absence of glycolysis and mitochondrial ATP synthesis, and is unique to cancer. Biochemical assays using viable cancer cells, and purified plasma membrane vesicles utilizing radioactive phosphate, confirmed phosphate-bond synthesis from free phosphate (Pi), and also localization of this activity to the plasma membrane. In addition to ATP, predominant formation of pyrophosphate (PPi) from Pi was also observed when plasma membrane vesicles from cancer cells were subjected to trans-membrane acid gradient. Cancer cytosols were found capable of converting PPi to ATP, and also stimulate ATP synthesis from Pi from the vesicles. Acid gradient created through glucose metabolism by cancer cells, as observed in tumors, also proved critical for phosphate-bond synthesis. In brief, these observations reveal a role of acidic tumor milieu as a potential energy source and may offer a novel therapeutic target.

Cystathionine beta-synthase plays a key role in the intracellular disposal of homocysteine and is the single most common locus of mutations associated with homocystinuria. Elevated levels of homocysteine are correlated with heart disease, Alzheimer's and Parkinson's diseases, and neural tube defects. Cystathionine beta-synthase is modular and subjected to complex regulation, but insights into the structural basis of this regulation are lacking. We have employed hydrogen exchange mass spectrometry to map peptides whose motions are correlated with transmission of intrasteric inhibition and allosteric activation. The mass spectrometric data provide an excellent correlation between kinetically and conformationally distinguishable states of the enzyme. We also demonstrate that a pathogenic regulatory domain mutant, D444N, is conformationally locked in one of two states sampled by the wild type enzyme. Our hydrogen exchange data identify surfaces that are potentially involved in the juxtaposition of the regulatory and catalytic domains and form the basis of a docked structural model for the full-length enzyme.

Gestational diabetes, pre-eclampsia as well as intra-uterine infection during pregnancy affects the function of the endothelium both in the mother and the fetus leading to endothelial dysfunction. Gestational diabetes is also associated with an increased incidence of pre-eclampsia and it is likely that both the hyperglycemia as well as the release of cytokines especially TNFα during hyperglycemia may play an important role in the pathogenesis of endothelial dysfunction leading to preeclampsia. Similarly, some but not all studies have suggested that infection of the mother under certain circumstances can also lead to preeclampsia as women with either a bacterial or viral infection were at a higher risk of developing preeclampsia, compared to women without infection and infection also leads to a release in TNFα. Endothelial cells exposed to either high glucose or TNFα leads to an increase in the production of H2O2 and to a decrease in endothelial cell proliferation. The cellular and molecular mechanisms involved in this phenomenon are discussed.Gestational diabetes, pre-eclampsia as well as intra-uterine infection during pregnancy has profound effects on the fetus and long term effects on the neonate. All three conditions affect the function of the endothelium both in the mother and the fetus leading to endothelial dysfunction. Gestational diabetes is also associated with an increased incidence of pre-eclampsia and it is likely that both the hyperglycemia as well as the release of cytokines especially TNFα during hyperglycemia may play an important role in the pathogenesis of endothelial dysfunction leading to preeclampsia. It has also been suggested although not universally accepted that under certain circumstances maternal infection may also predispose to pre-eclampsia. Pre-eclampsia is also associated with the release of TNFα and endothelial dysfunction. However, the cellular and molecular mechanism(s) leading to the endothelial dysfunction by either hyperglycemia or by the cytokine TNFα appear to be different. In this chapter, we explore some of the similarities and differences leading to endothelial dysfunction by both hyperglycemia and by the inflammatory cytokine TNFα and the cellular and molecular mechanism(s) involved.

Gestational diabetes, pre-eclampsia as well as intra-uterine infection during pregnancy affects the function of the endothelium both in the mother and the fetus leading to endothelial dysfunction. Gestational diabetes is also associated with an increased incidence of pre-eclampsia and it is likely that both the hyperglycemia as well as the release of cytokines especially TNFα during hyperglycemia may play an important role in the pathogenesis of endothelial dysfunction leading to preeclampsia. Similarly, some but not all studies have suggested that infection of the mother under certain circumstances can also lead to preeclampsia as women with either a bacterial or viral infection were at a higher risk of developing preeclampsia, compared to women without infection and infection also leads to a release in TNFα. Endothelial cells exposed to either high glucose or TNFα leads to an increase in the production of H2O2 and to a decrease in endothelial cell proliferation. The cellular and molecular mechanisms involved in this phenomenon are discussed.

We have previously demonstrated that relatively high concentrations of NO [Nitric Oxide] as produced by activated macrophages induced apoptosis in the human breast cancer cell line, MDA-MB-468. More recently, we also demonstrated the importance of endogenous H2O2 in the regulation of growth in human breast cancer cells. In the present study we assessed the interplay between exogenously administered NO and the endogenously produced reactive oxygen species [ROS] in human breast cancer cells and evaluated the mechanism[s] in the induction of apoptosis. To this end we identified a novel mechanism by which NO down regulated endogenous hydrogen peroxide [H2O2] formation via the down-regulation of superoxide [O2 (.-)] and the activation of catalase. We further demonstrated the existence of a feed forward mechanistic loop involving protein phosphatase 2A [PP2A] and its downstream substrate FOXO1 in the induction of apoptosis and the synthesis of catalase. We utilized gene silencing of PP2A, FOXO1 and catalase to assess their relative importance and key roles in NO mediated apoptosis. This study provides the potential for a therapeutic approach in treating breast cancer by targeted delivery of NO where NO donors and activators of downstream players could initiate a self sustaining apoptotic cascade in breast cancer cells.

We assessed the catalase bioactivity and hydrogen peroxide (H₂O₂) production rate in human breast cancer (HBC) cell lines and compared these with normal human breast epithelial (HBE) cells. We observed that the bioactivity of catalase was decreased in HBC cells when compared with HBE cells. This was also accompanied by an increase in H₂O₂ steady-state levels in HBC cells. Silencing the catalase gene led to a further increase in the steady-state level of H₂O₂ which was also accompanied by an increase in growth rate of HBC cells. Catalase activity was up regulated on treatment with superoxide (O₂⁻) scavengers such as pegylated SOD (PEG-SOD, indicating inhibition of catalase by the increased O₂⁻ produced by HBC cells. Transfection of either catalase or glutathione peroxidase to HBC cells decreased intracellular H₂O₂ levels and led to apoptosis of these cells. The H₂O₂ produced by HBC cells inhibited PP2A activity accompanied by increased phosphorylation of Akt and ERK1/2. The importance of catalase bioactivity in breast cancer was further confirmed as its bioactivity was also decreased in human breast cancer tissues when compared to normal breast tissues. We conclude that inhibition of catalase bioactivity by O₂⁻ leads to an increase in steady-state levels of H₂O₂ in HBC cells, which in turn inhibits PP2A activity, leading to phosphorylation of ERK 1/2 and Akt and resulting in HBC cell proliferation.

Nitric oxide (NO) is produced and nitric oxide synthase (NOS) activity is expressed in many types of tumor cells, but their precise role in tumor proliferation has not been clearly elucidated. Recently, it has been observed that patients with triple-negative breast tumors expressing NOS have a significantly worse prognosis compared to those that do not express any NOS. We observed that NOS activity was associated with the mitochondria in two breast cancer cell lines, ZR-75-30 and BT-474, compared with another NO-producing benign breast epithelial cell line, MCF-12F, in which no significant mitochondrial-associated NOS activity was detected. The rate of proliferation of the malignant cells expressing mitochondrial-associated NOS was decreased in the presence of an inhibitor of NO synthesis, but it had no effect on the normal breast epithelial cells, MCF-12F, which also expressed NOS, but not associated with mitochondria. The basal rate of proliferation was not affected by ODQ, an inhibitor of soluble guanylate cyclase, indicating that the effects of the endogenous NO produced by the malignant cell lines on proliferation are cGMP independent. Our results indicate that mitochondrial-associated NOS activity exhibited by the cancer cell lines ZR-75-30 and BT-474 inhibited cytochrome c oxidase, resulting in increased production of hydrogen peroxide (H2O2), which inhibited protein phosphatase 2A activity. This resulted in the maintenance of Akt and ERK1/2 in a phosphorylated state, leading to cell proliferation.

Extensive research work performed over the past decades has focused on understanding the role of nitric oxide (NO) in both promoting and preventing cancer. The precise role of NO in tumor biology has been the cause of intense debate. Experimental evidences available in the literature highlight contrasting pro- and anti-tumor effects of NO. It is now becoming clear that concentration- and time-dependent regulation of NO leads to tumor growth, cytostasis, or cell death. It is known that NO participates in various signaling pathways including Ras, extracellular signal-regulated kinases (ERKs), Akt, cyclin D1/retinoblastoma (Rb) as mammalian target of rapamycin (mTOR) that are crucial for tumor cells. NO mediated post-translational modification of key proteins including S-nitrosylation of caspases, tyrosine nitration of mitochondrial manganese superoxide dismutase (MnSOD), or cytochrome c. Various attempts toward developing NO-based cancer therapy are still in primitive stages, and a clear understanding of the levels of NOS expression, its timing, and the concentrations of NO produced in the tumor microenvironment is key to the development of novel strategies for tumor treatment and prevention.

Human cystathionine beta-synthase plays a key role in maintaining low intracellular levels of homocysteine and is unique in being a pyridoxal phosphate-dependent enzyme that is a hemeprotein. It catalyzes the beta-replacement of serine and homocysteine to generate the condensation product, cystathionine. While the structure of a truncated catalytic core of the protein has been determined by crystallography, a model for the full-length enzyme has been developed guided by hydrogen-deuterium exchange mass spectrometric and docking studies. In this review, we have utilized the available structural models for human cystathionine beta-synthase to conduct a structure-function analysis of a select group of pathogenic mutations described in patients with hereditary hyperhomocysteinemia.