Y chromosome loss in cancer drives growth by evasion of adaptive immunity.

Loss of the Y chromosome (LOY) is observed in multiple cancer types, including 10-40% of bladder cancers1-6, but its clinical and biological significance is unknown. Here, using genomic and transcriptomic studies, we report that LOY correlates with poor prognoses in patients with bladder cancer. We performed in-depth studies of naturally occurring LOY mutant bladder cancer cells as well as those with targeted deletion of Y chromosome by CRISPR-Cas9. Y-positive (Y+) and Y-negative (Y-) tumours grew similarly in vitro, whereas Y- tumours were more aggressive than Y+ tumours in immune-competent hosts in a T cell-dependent manner. High-dimensional flow cytometric analyses demonstrated that Y- tumours promote striking dysfunction or exhaustion of CD8+ T cells in the tumour microenvironment. These findings were validated using single-nuclei RNA sequencing and spatial proteomic evaluation of human bladder cancers. Of note, compared with Y+ tumours, Y- tumours exhibited an increased response to anti-PD-1 immune checkpoint blockade therapy in both mice and patients with cancer. Together, these results demonstrate that cancer cells with LOY mutations alter T cell function, promoting T cell exhaustion and sensitizing them to PD-1-targeted immunotherapy. This work provides insights into the basic biology of LOY mutation and potential biomarkers for improving cancer immunotherapy.

TRIM28 is a transcriptional activator of the mutant TERT promoter in human bladder cancer.

Bladder cancer (BC) has a 70% telomerase reverse transcriptase (TERT or hTERT in humans) promoter mutation prevalence, commonly at -124 base pairs, and this is associated with increased hTERT expression and poor patient prognosis. We inserted a green fluorescent protein (GFP) tag in the mutant hTERT promoter allele to create BC cells expressing an hTERT-GFP fusion protein. These cells were used in a fluorescence-activated cell sorting-based pooled CRISPR-Cas9 Kinome knockout genetic screen to identify tripartite motif containing 28 (TRIM28) and TRIM24 as regulators of hTERT expression. TRIM28 activates, while TRIM24 suppresses, hTERT transcription from the mutated promoter allele. TRIM28 is recruited to the mutant promoter where it interacts with TRIM24, which inhibits its activity. Phosphorylation of TRIM28 through the mTOR complex 1 (mTORC1) releases it from TRIM24 and induces hTERT transcription. TRIM28 expression promotes in vitro and in vivo BC cell growth and stratifies BC patient outcome. mTORC1 inhibition with rapamycin analog Ridaforolimus suppresses TRIM28 phosphorylation, hTERT expression, and cell viability. This study may lead to hTERT-directed cancer therapies with reduced effects on normal progenitor cells.

Clinical and Preclinical Therapies for Bladder Cancer Following Bacillus Calmette-Guérin Failure.

PURPOSE: Intravesical bacillus Calmette-Guérin is the current first-line treatment for high-grade nonmuscle-invasive bladder cancer; however, a substantial proportion of patients are unresponsive to bacillus Calmette-Guérin treatment. While cystectomy is often recommended in bladder cancer following bacillus Calmette-Guérin failure, there are numerous established therapeutic agents and pre-commercialized trials describing treatments for nonmuscle-invasive bladder cancer following failed bacillus Calmette-Guérin treatment. Our objective in this systematic review is to characterize the efficacy of these therapeutic agents by reporting their corresponding complete response rates and toxicity profiles. MATERIALS AND METHODS: We conducted a systematic review of all available clinical trials evaluating therapies to treat recurring nonmuscle-invasive bladder cancer after previous intravesical bacillus Calmette-Guérin. Bacillus Calmette-Guérin failure patients who had previously failed 1 or more courses of prior bacillus Calmette-Guérin therapy were included. Studies that were not in the English language, included muscle-invasive bladder cancer patient populations, or lacked a post-treatment evaluation of response were excluded. We used PubMed/Medline, the Cochrane Library, and Embase to search for relevant studies. No formal risk of bias assessment was conducted. Complete response rates for 3, 6, 12, and 24 months post-treatment evaluation, progression rates, cystectomy rates, and 12 complications are reported. RESULTS: A total of 70 studies with 73 reports evaluating 27 treatment options were retained for final analysis. These treatments were reported in 5 categories including intravesical chemotherapy, combination therapy, hyperthermia paired with intravesical chemotherapy, immunotherapy, and novel agents, with published years ranging from 1998 to 2021. Single intravesical chemotherapy and the combination of multiple intravesical chemotherapy agents demonstrate varied complete response rates of 10%-83% at 12 months. Limited clinical data evaluating hyperthermia paired with chemotherapy demonstrate 12-month complete response rates of 50%-85%. Despite these reported response rates, progression rates ranged from 0%-18%. Moreover, immunotherapeutic agents demonstrate progression rates of 7% to 22% at a median of 12 months of follow-up. Novel agents displayed a wide range of complete response rates (6% to 91%) at 12 months based on the treatment used. Total grade 3 toxicity rates range from 0%-55% for intravesical chemotherapy and combination intravesical chemotherapy agents, 0%-15% for hyperthermia paired with chemotherapy agents, 12%-13% for immunotherapy agents, and 0%-17% for novel agents. CONCLUSIONS: Bladder-preserving treatments accomplish moderate success in nonmuscle-invasive bladder cancer following bacillus Calmette-Guérin failure. As the majority of available clinical trials are single-armed uncontrolled cohorts and contain a limited number of patients, strength and comparability of the data are limited. In general, intravesical chemotherapy and hyperthermia paired with mitomycin C demonstrate some of the highest complete response rates at 12 and 24 months. Similarly, among the pre-commercialized novel agents, N-803 and gene therapy display promising results and may serve as potential future treatment for nonmuscle-invasive bladder cancer following failed bacillus Calmette-Guérin treatment. In terms of toxicity/complication rates, both commercially available and unavailable treatments showcase low toxicity profiles for bladder cancer following bacillus Calmette-Guérin failure. The comprehensive analysis provided by this systematic review can serve as a reference for treatment decisions and clinical trial design in the bacillus Calmette-Guérin-unresponsive domain.

Mutation of the TERT promoter, switch to active chromatin, and monoallelic TERT expression in multiple cancers.

Somatic mutations in the promoter of the gene for telomerase reverse transcriptase (TERT) are the most common noncoding mutations in cancer. They are thought to activate telomerase, contributing to proliferative immortality, but the molecular events driving TERT activation are largely unknown. We observed in multiple cancer cell lines that mutant TERT promoters exhibit the H3K4me2/3 mark of active chromatin and recruit the GABPA/B1 transcription factor, while the wild-type allele retains the H3K27me3 mark of epigenetic silencing; only the mutant promoters are transcriptionally active. These results suggest how a single-base-pair mutation can cause a dramatic epigenetic switch and monoallelic expression.

AST-487 Inhibits RET Kinase Driven TERT Expression in Bladder Cancer.

Mutations in the promoter of the human Telomerase Reverse Transcriptase (hTERT) gene are common and associated with its elevated expression in bladder cancer, melanoma, and glioblastoma. Though these mutations and TERT overexpression are associated with aggressive disease and poor outcome, an incomplete understanding of mutant TERT regulation limits treatment options directed at this gene. Herein, we unravel a signaling pathway that leads to upregulated hTERT expression resulting from the -124 bp promoter mutation, the most frequent variant across human cancer. We employed engineered bladder cancer cells that harbor a GFP insertion at the TSS region on -124 hTERT promoter for high-content screening drug discovery using a focused library of ~800 kinase inhibitors. Studies using in vitro and in vivo models prioritized AST-487, an inhibitor of the wild-type, and mutant RET (rearranged during transfection) proto-oncogene as a novel drug inhibitor of both wild-type and mutant promoter-driven hTERT expression. We also identified the RET kinase pathway, targeted by AST-487, as a novel regulator of mutant hTERT promoter-driven transcription in bladder cancer cells. Collectively, our work provides new potential precision medicine approaches for cancer patients with upregulated hTERT expression, perhaps, especially those harboring mutations in both the RET gene and the hTERT promoter, such as in thyroid cancer.

High-Content Drug Discovery Targeting Molecular Bladder Cancer Subtypes.

Molecular subtypes of muscle-invasive bladder cancer (MIBC) display differential survival and drug sensitivities in clinical trials. To date, they have not been used as a paradigm for phenotypic drug discovery. This study aimed to discover novel subtype-stratified therapy approaches based on high-content screening (HCS) drug discovery. Transcriptome expression data of CCLE and BLA-40 cell lines were used for molecular subtype assignment in basal, luminal, and mesenchymal-like cell lines. Two independent HCSs, using focused compound libraries, were conducted to identify subtype-specific drug leads. We correlated lead drug sensitivity data with functional genomics, regulon analysis, and in-vitro drug response-based enrichment analysis. The basal MIBC subtype displayed sensitivity to HDAC and CHK inhibitors, while the luminal subtype was sensitive to MDM2 inhibitors. The mesenchymal-like cell lines were exclusively sensitive to the ITGAV inhibitor SB273005. The role of integrins within this mesenchymal-like MIBC subtype was confirmed via its regulon activity and gene essentiality based on CRISPR-Cas9 knock-out data. Patients with high ITGAV expression showed a significant decrease in the median overall survival. Phenotypic high-content drug screens based on bladder cancer cell lines provide rationales for novel stratified therapeutic approaches as a framework for further prospective validation in clinical trials.

The impact of the social construct of race on outcomes among bacille Calmette-Guérin-treated patients with high-risk non-muscle-invasive bladder cancer in an equal-access setting.

BACKGROUND: The objective of this study was to describe bladder cancer outcomes as a function of race among patients with high-risk non-muscle-invasive bladder cancer (NMIBC) in an equal-access setting. METHODS: A total of 412 patients with high-risk NMIBC who received bacille Calmette-Guérin (BCG) from January 1, 2010, to December 31, 2015, were assessed. The authors used the Kaplan-Meier method to estimate event-free survival and Cox regression to determine the association between race and recurrence, progression, disease-specific, and overall survival outcomes. RESULTS: A total of 372 patients who had complete data were included in the analysis; 48 (13%) and 324 (87%) were Black and White, respectively. There was no difference in age, sex, smoking status, or Charlson Comorbidity Index by race. White patients had a higher socioeconomic status with a greater percentage of patients living above the poverty level in comparison with Black patients (median, 85% vs 77%; P < .001). A total of 360 patients (97%) received adequate induction BCG, and 145 patients (39%) received adequate maintenance BCG therapy. There was no significant difference in rates of adequate induction or maintenance BCG therapy according to race. There was no significant difference in recurrence (hazard ratio [HR], 1.53; 95% confidence interval [CI], 0.64-3.63), progression (HR, 0.77; 95% CI, 0.33-1.82), bladder cancer-specific survival (HR, 1.01; 95% CI, 0.30-3.46), or overall survival (HR, 0.97; 95% CI, 0.56-1.66) according to Black race versus White race. CONCLUSIONS: In this small study from an equal-access setting, there was no difference in the receipt of BCG or any differences in bladder cancer outcomes according to race.

Metastatic cells are preferentially vulnerable to lysosomal inhibition.

Molecular alterations that confer phenotypic advantages to tumors can also expose specific therapeutic vulnerabilities. To search for potential treatments that would selectively affect metastatic cells, we examined the sensitivity of lineage-related human bladder cancer cell lines with different lung colonization abilities to chloroquine (CQ) or bafilomycin A1, which are inhibitors of lysosome function and autophagy. Both CQ and bafilomycin A1 were more cytotoxic in vitro to highly metastatic cells compared with their less metastatic counterparts. Genetic inactivation of macroautophagy regulators and lysosomal proteins indicated that this was due to greater reliance on the lysosome but not upon macroautophagy. To identify the mechanism underlying these effects, we generated cells resistant to CQ in vitro. Surprisingly, selection for in vitro CQ resistance was sufficient to alter gene expression patterns such that unsupervised cluster analysis of whole-transcriptome data indicated that selection for CQ resistance alone created tumor cells that were more similar to the poorly metastatic parental cells from which the metastatic cells were derived; importantly, these tumor cells also had diminished metastatic ability in vivo. These effects were mediated in part by differential expression of the transcriptional regulator ID4 (inhibitor of DNA binding 4); depletion of ID4 both promoted in vitro CQ sensitivity and restored lung colonization and metastasis of CQ-resistant cells. These data demonstrate that selection for metastasis ability confers selective vulnerability to lysosomal inhibitors and identify ID4 as a potential biomarker for the use of lysosomal inhibitors to reduce metastasis in patients.

RalA controls glucose homeostasis by regulating glucose uptake in brown fat.

Insulin increases glucose uptake into adipose tissue and muscle by increasing trafficking of the glucose transporter Glut4. In cultured adipocytes, the exocytosis of Glut4 relies on activation of the small G protein RalA by insulin, via inhibition of its GTPase activating complex RalGAP. Here, we evaluate the role of RalA in glucose uptake in vivo with specific chemical inhibitors and by generation of mice with adipocyte-specific knockout of RalGAPB. RalA was profoundly activated in brown adipose tissue after feeding, and its inhibition prevented Glut4 exocytosis. RalGAPB knockout mice with diet-induced obesity were protected from the development of metabolic disease due to increased glucose uptake into brown fat. Thus, RalA plays a crucial role in glucose transport in adipose tissue in vivo.

RAL GTPases: Biology and Potential as Therapeutic Targets in Cancer.

More than a hundred proteins comprise the RAS superfamily of small GTPases. This family can be divided into RAS, RHO, RAB, RAN, ARF, and RAD subfamilies, with each shown to play distinct roles in human cells in both health and disease. The RAS subfamily has a well-established role in human cancer with the three genes, HRAS, KRAS, and NRAS being the commonly mutated in tumors. These RAS mutations, most often functionally activating, are especially common in pancreatic, lung, and colorectal cancers. Efforts to inhibit RAS and related GTPases have produced inhibitors targeting the downstream effectors of RAS signaling, including inhibitors of the RAF-mitogen-activated protein kinase/extracellular signal-related kinase (ERK)-ERK kinase pathway and the phosphoinositide-3-kinase-AKT-mTOR kinase pathway. A third effector arm of RAS signaling, mediated by RAL (RAS like) has emerged in recent years as a critical driver of RAS oncogenic signaling and has not been targeted until recently. RAL belongs to the RAS branch of the RAS superfamily and shares a high structural similarity with RAS. In human cells, there are two genes, RALA and RALB, both of which have been shown to play roles in the proliferation, survival, and metastasis of a variety of human cancers, including lung, colon, pancreatic, prostate, skin, and bladder cancers. In this review, we summarize the latest knowledge of RAL in the context of human cancer and the recent advancements in the development of cancer therapeutics targeting RAL small GTPases.

Improving Anti-PD-1/PD-L1 Therapy for Localized Bladder Cancer.

In high-risk non-muscle invasive bladder cancer (HR-NMIBC), patient outcome is negatively affected by lack of response to Bacillus-Calmette Guérin (BCG) treatment. Lack of response to cisplatin-based neoadjuvant chemotherapy and cisplatin ineligibility reduces successful treatment outcomes in muscle-invasive bladder cancer (MIBC) patients. The effectiveness of PD-1/PD-L1 immune checkpoint inhibitors (ICI) in metastatic disease has stimulated its evaluation as a treatment option in HR-NMIBC and MIBC patients. However, the observed responses, immune-related adverse events and high costs associated with ICI have provided impetus for the development of methods to improve patient stratification, enhance anti-tumorigenic effects and reduce toxicity. Here, we review the challenges and opportunities offered by PD-1/PD-L1 inhibition in HR-NMIBC and MIBC. We highlight the gaps in the field that need to be addressed to improve patient outcome including biomarkers for response stratification and potentially synergistic combination therapy regimens with PD-1/PD-L1 blockade.

Discovery and characterization of small molecules that target the GTPase Ral.

The Ras-like GTPases RalA and RalB are important drivers of tumour growth and metastasis. Chemicals that block Ral function would be valuable as research tools and for cancer therapeutics. Here we used protein structure analysis and virtual screening to identify drug-like molecules that bind to a site on the GDP-bound form of Ral. The compounds RBC6, RBC8 and RBC10 inhibited the binding of Ral to its effector RALBP1, as well as inhibiting Ral-mediated cell spreading of murine embryonic fibroblasts and anchorage-independent growth of human cancer cell lines. The binding of the RBC8 derivative BQU57 to RalB was confirmed by isothermal titration calorimetry, surface plasmon resonance and (1)H-(15)N transverse relaxation-optimized spectroscopy (TROSY) NMR spectroscopy. RBC8 and BQU57 show selectivity for Ral relative to the GTPases Ras and RhoA and inhibit tumour xenograft growth to a similar extent to the depletion of Ral using RNA interference. Our results show the utility of structure-based discovery for the development of therapeutics for Ral-dependent cancers.

Determinants of Resistance to Checkpoint Inhibitors.

The development of immune checkpoint inhibitors (ICIs) has drastically altered the landscape of cancer treatment. Since approval of the first ICI for the treatment of advanced melanoma in 2011, several therapeutic agents have been Food and Drug Administration (FDA)-approved for multiple cancers, and hundreds of clinical trials are currently ongoing. These antibodies disrupt T-cell inhibitory pathways established by tumor cells and thus re-activate the host's antitumor immune response. While successful in many cancers, several types remain relatively refractory to treatment or patients develop early recurrence. Hence, there is a great need to further elucidate mechanisms of resistant disease and determine novel, effective, and tolerable combination therapies to enhance efficacy of ICIs.

Elucidating the role of Agl in bladder carcinogenesis by generation and characterization of genetically engineered mice.

Amylo-α-1,6-glucosidase,4-α-glucanotransferase (AGL) is an enzyme primarily responsible for glycogen debranching. Germline mutations lead to glycogen storage disease type III (GSDIII). We recently found AGL to be a tumor suppressor in xenograft models of human bladder cancer (BC) and low levels of AGL expression in BC are associated with poor patient prognosis. However, the impact of low AGL expression on the susceptibility of normal bladder to carcinogenesis is unknown. We address this gap by developing a germline Agl knockout (Agl-/-) mouse that recapitulates biochemical and histological features of GSDIII. Agl-/- mice exposed to N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) had a higher BC incidence compared with wild-type mice (Agl+/+). To determine if the increased BC incidence observed was due to decreased Agl expression in the urothelium specifically, we developed a urothelium-specific conditional Agl knockout (Aglcko) mouse using a Uroplakin II-Cre allele. BBN-induced carcinogenesis experiments repeated in Aglcko mice revealed that Aglcko mice had a higher BC incidence than control (Aglfl/fl) mice. RNA sequencing revealed that tumors from Agl-/- mice had 19 differentially expressed genes compared with control mice. An 'Agl Loss' gene signature was developed and found to successfully stratify normal and tumor samples in two BC patient datasets. These results support the role of AGL loss in promoting carcinogenesis and provide a rationale for evaluating Agl expression levels, or Agl Loss gene signature scores, in normal urothelium of populations at risk of BC development such as older male smokers.

Assessment of roles for the Rho-specific guanine nucleotide dissociation inhibitor Ly-GDI in platelet function: a spatial systems approach.

On activation at sites of vascular injury, platelets undergo morphological alterations essential to hemostasis via cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42, and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in platelet function. We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. Whereas RhoGDI localizes throughout platelets in a granule-like manner, Ly-GDI shows an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI, which is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP) VI-specific agonist collagen-related peptide. Additionally, PKC inhibition diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Together, our results suggest a role for Ly-GDI in the localized regulation of Rho GTPases in platelets and hypothesize a link between the PKC and Rho GTPase signaling systems in platelet function.

A rho GDP dissociation inhibitor produced by apoptotic T-cells inhibits growth of Mycobacterium tuberculosis.

In this study, we found that a subpopulation of CD4(+)CD25(+) (85% Foxp3(+)) cells from persons with latent tuberculosis infection (LTBI) inhibits growth of M. tuberculosis (M. tb) in human monocyte-derived macrophages (MDMs). A soluble factor, Rho GDP dissociation inhibitor (D4GDI), produced by apoptotic CD4(+)CD25(+) (85% Foxp3(+)) cells is responsible for this inhibition of M. tb growth in human macrophages and in mice. M. tb-expanded CD4(+C)D25(+)Foxp3(+)D4GDI(+) cells do not produce IL-10, TGF-ß and IFN-γ. D4GDI inhibited growth of M. tb in MDMs by enhancing production of IL-1ß, TNF-α and ROS, and by increasing apoptosis of M. tb-infected MDMs. D4GDI was concentrated at the site of disease in tuberculosis patients, with higher levels detected in pleural fluid than in serum. However, in response to M. tb, PBMC from tuberculosis patients produced less D4GDI than PBMC from persons with LTBI. M. tb-expanded CD4+CD25+ (85% Foxp3(+)) cells and D4GDI induced intracellular M. tb to express the dormancy survival regulator DosR and DosR-dependent genes, suggesting that D4GDI induces a non-replicating state in the pathogen. Our study provides the first evidence that a subpopulation of CD4(+)CD25(+) (85% Foxp3+) cells enhances immunity to M. tb, and that production of D4GDI by this subpopulation inhibits M. tb growth.

Clinical Decision Making in Surveillance of Non-Muscle-Invasive Bladder Cancer: The Evolving Roles of Urinary Cytology and Molecular Markers.

Cystoscopy and urine cytology are the gold-standard tests for detection of recurrent disease during follow-up in patients with a history of non-muscle-invasive bladder cancer (NMIBC). High associated costs, as well as side effects, have driven the desire for inexpensive, noninvasive, accurate, and easy-to-use urine markers to detect bladder cancer recurrence. While many urine markers have been developed, very few have been clinically implemented. In this article, we discuss the requirements for development and validation of urine markers and the factors that hamper their clinical implementation. We also review current surveillance guidelines for NMIBC and provide an overview of approved urine markers for the detection and surveillance of NMIBC.

CD44 and RHAMM are essential for rapid growth of bladder cancer driven by loss of Glycogen Debranching Enzyme (AGL).

BACKGROUND: Loss of Amylo-alpha-1-6-glucosidase-4-alpha-glucanotransferase (AGL) drives rapid proliferation of bladder cancer cells by upregulating Hyaluronic acid(HA) Synthase (HAS2) mediated HA synthesis. However the role of HA receptors CD44 and Hyaluronan Mediated Motility Receptor (RHAMM) in regulating the growth of bladder cancer cells driven by loss of AGL has not been studied. METHODS: Western blot analysis and Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was carried out to study cellular apoptosis with HAS2, CD44 and RHAMM loss in bladder cancer cells with and without AGL expression. Proliferation and softagar assays were carried out to study cellular anchorage dependent and independent growth. Clinicopathologic analysis was carried out on bladder cancer patient datasets. RESULTS: Higher amounts of cleaved Cas3, Cas9 and PARP was observed in AGL low bladder cancer cell with loss of HAS2, CD44 or RHAMM. TUNEL staining showed more apoptotic cells with loss of HAS2, CD44 or RHAMM in AGL low bladder cancer cells. This revealed that bladder cancer cells whose aggressive growth is mediated by loss of AGL are susceptible to apoptosis with loss of HAS2, CD44 or RHAMM. Interestingly loss of either CD44 or RHAMM induces apoptosis in different low AGL expressing bladder cancer cell lines. Growth assays showed that loss of CD44 and RHAMM predominantly inhibit anchorage dependent and independent growth of AGL low bladder cancer cells. Clinicopathologic analysis revealed that high RHAMM mRNA expression is a marker of poor patient outcome in bladder cancer and patients with high RHAMM and low AGL tumor mRNA expression have poor survival. CONCLUSION: Our findings strongly point to the importance of the HAS2-HA-CD44/RHAMM pathway for rapid growth of bladder cancer cells with loss of AGL and provides rational for targeting this pathway at various steps for "personalized" treatment of bladder cancer patients based of their AGL expression status.

Synthesis of novel Ral inhibitors: An in vitro and in vivo study.

Chemical synthesis was performed to produce a series of 6-amino-1,3-disubstituted-4-phenyl-1,4-dihydro pyrano[2,3-c]pyrazole-5-carbonitrile compounds (14-57) which were characterized by 1H NMR, 13C NMR and LC/MS-MS. These compounds were assessed for their effect on the in vitro anchorage independent growth of human lung cancer cell line H2122 and IC50 values calculated. Two of the more potent compounds, BQU057 40 and BQU082 57 also displayed a dose dependent effect on RalA and RalB activity in H2122 spheroids using the common RalBP1 pull-down assay. Mouse PK and tissue distribution studies on 40 and 57 were performed and demonstrated that parent drug was present in tumor 3.0h post ip (50mg/Kg) dose.