Development of substituted benzimidazoles as inhibitors of human aldehyde dehydrogenase 1A isoenzymes.

Aldehyde dehydrogenase 1A (ALDH1A) isoforms may be a useful target for overcoming chemotherapy resistance in high-grade serous ovarian cancer (HGSOC) and other solid tumor cancers. However, as different cancers express different ALDH1A isoforms, isoform selective inhibitors may have a limited therapeutic scope. Furthermore, resistance to an ALDH1A isoform selective inhibitor could arise via induction of expression of other ALDH1A isoforms. As such, we have focused on the development of pan-ALDH1A inhibitors, rather than on ALDH1A isoform selective compounds. Herein, we report the development of a new group of pan-ALDH1A inhibitors to assess whether broad spectrum ALDH1A inhibition is an effective adjunct to chemotherapy in HGSOC. Optimization of the CM10 scaffold, aided by ALDH1A1 crystal structures, led to improved biochemical potencies, improved cellular efficacy as demonstrated by reduction in ALDEFLUOR signal in HGSOC cells, and substantial improvements in liver microsomal stability. Based on this work we identified two compounds 17 and 25 suitable for future in vivo proof of concept experiments.

A putative role for ALDH inhibitors and chemoprevention of BRCA-mutation-driven tumors.

Aldehyde dehydrogenase (ALDH) enzymatic activity is a marker of cancer-initiating cells (CIC) in many tumor types. Our group and others have found that ALDH1A family inhibitors (ALDHi) can preferentially induce death of ovarian CIC in established ovarian cancer. We sought to determine if ALDHi, by targeting CIC at the time of tumor initiation, could function as a chemopreventive for ovarian cancer. As BRCA1/2 mutation carriers represent a population who could benefit from an ovarian cancer chemopreventive, we focused on BRCA mutation-associated tumor cell lines and murine tumor models. We found that, compared to BRCA wild-type cells, BRCA mutant ovarian cancer cells are more sensitive to the ALDHi673A. Similarly, while 673A treatment of wild-type fallopian tube epithelial (FTE) cells is non-toxic, 673A induces death in FTE cells with BRCA1 knockdown. Using a murine fallopian tube organoid model of ovarian carcinogenesis, we show that 673A reduced organoid complexity and significantly reduce colony formation of BRCA-mutant cells. Organoids that persisted after 673A treatment were predominantly BRCA1wt, but NF1 mutant, suggesting a resistance mechanism. Finally, using the BPRN (Brca1, Trp53, Rb1, Nf1 inactivated) mouse model of tubo-ovarian cancer, we evaluated the impact of intermittent 673A therapy on carcinogenesis. 673A treatment resulted in a significant reduction in serous tubal intraepithelial carcinoma (STIC) lesions and carcinomas. Collectively, the findings suggest that ALDHi, such as 673A, could serve as chemopreventive agents for BRCA1/2 mutation carriers.

A Novel Humanized Immune Stroma PDX Cancer Model for Therapeutic Studies.

Standard preclinical human tumor models lack a human tumor stroma. However, as stroma contributes to therapeutic resistance, the lack of human stroma may make current models less stringent for testing new therapies. To address this, using patient-derived tumor cells, patient derived cancer-associated mesenchymal stem/progenitor cells, and human endothelial cells, we created a Human Stroma-Patient Derived Xenograft (HS-PDX) tumor model. HS-PDX, compared to the standard PDX model, demonstrate greater resistance to targeted therapy and chemotherapy, and better reflect patient response to therapy. Furthermore, HS-PDX can be grown in mice with humanized bone marrow to create humanized immune stroma patient-derived xenograft (HIS-PDX) models. The HIS-PDX model contains human connective tissues, vascular and immune cell infiltrates. RNA sequencing analysis demonstrated a 94-96% correlation with primary human tumor. Using this model, we demonstrate the impact of human tumor stroma on response to CAR-T cell therapy and immune checkpoint inhibitor therapy. We show an immunosuppressive role for human tumor stroma and that this model can be used to identify immunotherapeutic combinations to overcome stromally mediated immunosuppression. Combined, our data confirm a critical role for human stoma in therapeutic response and indicate that HIS-PDX can be an important tool for preclinical drug testing. Statement of Significance: We developed a tumor model with human stromal, vascular, and immune cells. This model mirrors patient response to chemotherapy, targeted therapy, and immunotherapy, and can be used to study therapy resistance.

Targeting Therapeutic Resistance and Multinucleate Giant Cells in CCNE1-Amplified HR-Proficient Ovarian Cancer.

Approximately 20% of high-grade serous ovarian cancers (HGSOC) have CCNE1 amplification. CCNE1-amplified tumors are homologous recombination (HR) proficient and resistant to standard therapies. Therapy resistance is associated with increased numbers of polyploid giant cancer cells (PGCC). We sought to identify new therapeutic approaches for patients with CCNE1-amplified tumors. Using TCGA data, we find that the mTOR, HR, and DNA checkpoint pathways are enriched in CCNE1-amplified ovarian cancers. Furthermore, Interactome Mapping Analysis linked the mTOR activity with upregulation of HR and DNA checkpoint pathways. Indeed, we find that mTOR inhibitors (mTORi) downregulate HR/checkpoint genes in CCNE1-amplified tumors. As CCNE1-amplified tumors are dependent on the HR pathway for viability, mTORi proved selectively effective in CCNE1-amplified tumors. Similarly, via downregulation of HR genes, mTORi increased CCNE1-amplifed HGSOC response to PARPi. In contrast, overexpression of HR/checkpoint proteins (RAD51 or ATR), induced resistance to mTORi. In vivo, mTORi alone potently reduced CCNE1-amplified tumor growth and the combination of mTORi and PARPi increased response and tumor eradication. Tumors treated with mTORi demonstrated a significant reduction in ALDH+ PGCCs. Finally, as a proof of principle, we identified three patients with CCNE1 amplified tumors who were treated with an mTORi. All three obtained clinical benefits from the therapy. Our studies and clinical experience indicate mTORi are a potential therapeutic approach for patients with CCNE1-amplified tumors.

Aldehyde dehydrogenase inhibitors promote DNA damage in ovarian cancer and synergize with ATM/ATR inhibitors.

Rationale: Aldehyde dehydrogenase (ALDH) enzymes are often upregulated in cancer cells and associated with therapeutic resistance. ALDH enzymes protect cells by metabolizing toxic aldehydes which can induce DNA double stand breaks (DSB). We recently identified a novel ALDH1A family inhibitor (ALDHi), 673A. We hypothesized that 673A, via inhibition of ALDH1A family members, could induce intracellular accumulation of genotoxic aldehydes to cause DSB and that ALDHi could synergize with inhibitors of the ATM and ATR, proteins which direct DSB repair. Methods: We used immunofluorescence to directly assess levels of the aldehyde 4-hydroxynonenal and comet assays to evaluate DSB. Western blot was used to evaluate activation of the DNA damage response pathways. Cell counts were performed in the presence of 673A and additional aldehydes or aldehyde scavengers. ALDH inhibition results were confirmed using ALDH1A3 CRISPR knockout. Synergy between 673A and ATM or ATR inhibitors was evaluated using the Chou-Talalay method and confirmed in vivo using cell line xenograft tumor studies. Results: The ALDHi 673A cellular accumulation of toxic aldehydes which induce DNA double strand breaks. This is exacerbated by addition of exogenous aldehydes such as vitamin-A (retinaldehyde) and ameliorated by aldehyde scavengers such as metformin and hydralazine. Importantly, ALDH1A3 knockout cells demonstrated increased sensitivity to ATM/ATR inhibitors. And, ALDHi synergized with inhibitors of ATM and ATR, master regulators of the DSB DNA damage response, both in vitro and in vivo. This synergy was evident in homologous recombination (HR) proficient cell lines. Conclusions: ALDHi can be used to induce DNA DSB in cancer cells and synergize with inhibitors the ATM/ATR pathway. Our data suggest a novel therapeutic approach to target HR proficient ovarian cancer cells.

Development of 2,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one inhibitors of aldehyde dehydrogenase 1A (ALDH1A) as potential adjuncts to ovarian cancer chemotherapy.

There is strong evidence that inhibition of one or more Aldehyde Dehydrogenase 1A (ALDH1A) isoforms may be beneficial in chemotherapy-resistant ovarian cancer and other tumor types. While many previous efforts have focused on development of ALDH1A1 selective inhibitors, the most deadly ovarian cancer subtype, high-grade serous (HGSOC), exhibits elevated expression of ALDH1A3. Herein, we report continued development of pan-ALDH1A inhibitors to assess whether broad spectrum ALDH1A inhibition is an effective adjunct to chemotherapy in this critical tumor subtype. Optimization of the CM39 scaffold, aided by metabolite ID and several new ALDH1A1 crystal structures, led to improved biochemical potencies, improved cellular ALDH inhibition in HGSOC cell lines, and substantial improvements in microsomal stability culminating in orally bioavailable compounds. We demonstrate that two compounds 68 and 69 are able to synergize with chemotherapy in a resistant cell line and patient-derived HGSOC tumor spheroids, indicating their suitability for future in vivo proof of concept experiments.

Impairment of trophoblast survival and differentiation by LXR ligands is prevented by cholesterol but not ABCA1 silencing.

INTRODUCTION: The Liver X Receptors (LXRs) drive the transcriptional response to excess intracellular cholesterol. Oxysterols, the products of cholesterol oxidation, are activating ligands for LXR that can accumulate under conditions of oxidative stress and disrupt cholesterol homeostasis. While activation of LXR inhibits trophoblast differentiation, the impact of LXR on trophoblast physiology or cholesterol homeostasis is incompletely understood. We sought to determine if the effects of LXR activation can be ameliorated through modification of cholesterol bioavailability or inhibition of LXR-driven cholesterol efflux in trophoblasts. METHODS: We measured the effect of oxysterol exposure on BeWo cells and primary human trophoblasts (PHT cells) cultured in lipoprotein-deficient medium. We also measured the effect of the synthetic, LXR-specific ligand T0901317 on PHT cell differentiation and survival. Finally, we silenced the ATP-binding cassette transporter A1 (ABCA1), a transcriptional target of LXR that drives cholesterol efflux, to determine if inhibition of cholesterol efflux could block the effects of T0901317. RESULTS: Oxysterols inhibited BeWo survival and PHT cell differentiation, and these effects were blocked by cholesterol supplementation. T0901317 also inhibited PHT cell differentiation, and this effect was similarly blocked by cholesterol. Unlike cholesterol however, ABCA1 silencing did not modify the effect of T0901317 on PHT cell differentiation. DISCUSSION: Oxysterols and LXR inhibit trophoblast survival and differentiation exclusively in conditions of cholesterol scarcity. These findings underscore the importance of cholesterol homeostasis in the maintenance of placental function and suggest that pathways regulating cholesterol homeostasis may represent therapeutic targets to mitigate harmful sequelae of placental injury.

Low Soluble Syndecan-1 Precedes Preeclampsia.

INTRODUCTION: Syndecan-1 (Sdc1; CD138) is a major transmembrane heparan sulfate proteoglycan expressed on the extracellular, luminal surface of epithelial cells and syncytiotrophoblast, thus comprising a major component of the glycocalyx of these cells. The "soluble" (shed) form of Sdc1 has paracrine and autocrine functions and is normally produced in a regulated fashion. We compared plasma soluble Sdc1 concentrations, in relation to placental Sdc1 expression, in uncomplicated (control) and preeclamptic pregnancies. METHODS: We evaluated soluble Sdc1 across uncomplicated pregnancy, and between preeclamptic, gestational hypertensive and control patients at mid-pregnancy (20 weeks) and 3rd trimester by ELISA. Placental expression level of Sdc1 was compared between groups in relation to pre-delivery plasma soluble Sdc1. Participants were recruited from Magee-Womens Hospital. RESULTS: In uncomplicated pregnancy, plasma soluble Sdc1 rose significantly in the 1st trimester, and reached an approximate 50-fold increase at term compared to post pregnancy levels. Soluble Sdc1 was lower at mid-pregnancy in women who later developed preeclampsia (P<0.05), but not gestational hypertension, compared to controls, and remained lower at late pregnancy in preeclampsia (P<0.01) compared to controls. Sdc1 was prominently expressed on syncytiotrophoblast of microvilli. Syncytiotrophoblast Sdc1 immunostaining intensities, and mRNA content in villous homogenates, were lower in preeclampsia vs. controls (P<0.05). Soluble Sdc1 and Sdc1 immunostaining scores were inversely associated with systolic blood pressures, and positively correlated with infant birth weight percentile. CONCLUSION: Soluble Sdc1 is significantly lower before the clinical onset of preeclampsia, with reduced expression of Sdc1 in the delivered placenta, suggesting a role for glycocalyx disturbance in preeclampsia pathophysiology.

Cigarette exposure induces changes in maternal vascular function in a pregnant mouse model.

Smoking is associated with multiple adverse pregnancy outcomes, including fetal growth restriction. The objective of this study was to determine whether cigarette smoke exposure during pregnancy in a mouse model affects the functional properties of maternal uterine, mesenteric, and renal arteries as a possible mechanism for growth restriction. C57Bl/CJ mice were exposed to whole body sidestream smoke for 4 h/day. Smoke particle exposure was increased from day 4 of gestation until late pregnancy (day 16-19), with mean total suspended particle levels of 63 mg/m(3), representative of moderate-to-heavy smoking in humans. Uterine, mesenteric, and renal arteries from late-pregnant and virgin mice were isolated and studied in a pressure-arteriograph system (n = 23). Plasma cotinine was measured by ELISA. Fetal weights were significantly reduced in smoke-exposed compared with control fetuses (0.88 +/- 0.1 vs. 1.0 +/- 0.08 g, P < 0.02), while litter sizes were not different. Endothelium-mediated relaxation responses to methacholine were significantly impaired in both the uterine and mesenteric vasculature of pregnant mice exposed to cigarette smoke during gestation. This difference was not apparent in isolated renal arteries from pregnant mice exposed to cigarette smoke; however, relaxation was significantly reduced in renal arteries from smoke-exposed virgin mice. In conclusion, we found that passive cigarette smoke exposure is associated with impaired vascular relaxation of uterine and mesenteric arteries in pregnant mice. Functional maternal vascular perturbations during pregnancy, specifically impaired peripheral and uterine vasodilation, may contribute to a mechanism by which smoking results in fetal growth restriction.

Intracellular adhesion molecule concentrations in women who smoke during pregnancy.

OBJECTIVE: Smoking and endothelial dysfunction are associated with adverse pregnancy outcomes. The effect of smoking on vascular endothelium during pregnancy has not been well studied. Our objectives were to determine if smoking has an impact on endothelial function in pregnancy by comparing markers of endothelial function and to evaluate the contribution from different cellular sources. METHODS: We measured markers of endothelial function in a prospective cohort of 198 primiparous women who had 325 plasma samples obtained throughout pregnancy. Samples were assayed for intracellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin. Smoking status was determined by serum cotinine concentration. Analyses of adhesion molecules were performed for 4 gestational age intervals by using Mann-Whitney and Kruskal-Wallis tests. Gene expression for ICAM-1 was determined by real-time polymerase chain reaction from placental biopsies. A human umbilical vein endothelial cell (HUVEC) culture model was utilized to evaluate the effect of cotinine on endothelial cell production of ICAM-1. RESULTS: ICAM-1 is increased, VCAM-1 was not different, and E-selectin was decreased among smokers at various times during pregnancy. Placental production of ICAM-1 was decreased in women who smoked (P = .02) as measured by real-time polymerase chain reaction. Human umbilical vein endothelial cells production of ICAM-1 increased with heavy concentrations of cotinine exposure (P < .01). CONCLUSION: Smoking during pregnancy is associated with vascular perturbations, as evidenced by increased concentrations of serum ICAM-1. It appears unlikely that the source of the increased ICAM-1 is the placenta. The endothelium most likely contributes to increased maternal ICAM-1 in heavy smokers, but a leukocyte source cannot be ruled out. LEVEL OF EVIDENCE: II-2.