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KRAS inhibitors demonstrate clinical efficacy in pancreatic ductal adenocarcinoma (PDAC); however, resistance is common. Among patients with KRASG12C-mutant PDAC treated with adagrasib or sotorasib, mutations in PIK3CA and KRAS, and amplifications of KRASG12C, MYC, MET, EGFR, and CDK6 emerged at acquired resistance. In PDAC cell lines and organoid models treated with the KRASG12D inhibitor MRTX1133, epithelial-to-mesenchymal transition and PI3K-AKT-mTOR signaling associate with resistance to therapy. MRTX1133 treatment of the KrasLSL-G12D/+; Trp53LSL-R172H/+; p48-Cre (KPC) mouse model yielded deep tumor regressions, but drug resistance ultimately emerged, accompanied by amplifications of Kras, Yap1, Myc, Cdk6, and Abcb1a/b, and co-evolution of drug-resistant transcriptional programs. Moreover, in KPC and PDX models, mesenchymal and basal-like cell states displayed increased response to KRAS inhibition compared to the classical state. Combination treatment with KRASG12D inhibition and chemotherapy significantly improved tumor control in PDAC mouse models. Collectively, these data elucidate co-evolving resistance mechanisms to KRAS inhibition and support multiple combination therapy strategies. Significance: Acquired resistance may limit the impact of KRAS inhibition in patients with PDAC. Using clinical samples and multiple preclinical models, we define heterogeneous genetic and non-genetic mechanisms of resistance to KRAS inhibition that may guide combination therapy approaches to improve the efficacy and durability of these promising therapies for patients. See related commentary by Marasco and Misale, p. 2018., (©2024 The Authors; Published by the American Association for Cancer Research.)

Adagrasib, an irreversible, selective KRASG12C inhibitor, may be an effective treatment in KRASG12C-mutated colorectal cancer, particularly when combined with an anti-EGFR antibody. In this analysis of the KRYSTAL-1 trial, patients with previously treated KRASG12C-mutated unresectable or metastatic colorectal cancer received adagrasib (600 mg twice daily) plus cetuximab. The primary endpoint was objective response rate (ORR) by blinded independent central review. Ninety-four patients received adagrasib plus cetuximab. With a median follow-up of 11.9 months, ORR was 34.0%, disease control rate was 85.1%, and median duration of response was 5.8 months (95% confidence interval [CI], 4.2-7.6). Median progression-free survival was 6.9 months (95% CI, 5.7-7.4) and median overall survival was 15.9 months (95% CI, 11.8-18.8). Treatment-related adverse events (TRAEs) occurred in all patients; grade 3-4 in 27.7% and no grade 5. No TRAEs led to adagrasib discontinuation. Exploratory analyses suggest circulating tumor DNA may identify features of response and acquired resistance., Significance: Adagrasib plus cetuximab demonstrates promising clinical activity and tolerable safety in heavily pretreated patients with unresectable or metastatic KRASG12C-mutated colorectal cancer. These data support a potential new standard of care and highlight the significance of testing and identification of KRASG12C mutations in patients with colorectal cancer. This article is featured in Selected Articles from This Issue, p. 897., (©2024 The Authors; Published by the American Association for Cancer Research.)

Purpose: Non-invasive monitoring of circulating tumor DNA (ctDNA) has the potential to be a readily available measure for early prediction of clinical response. Here, we report on early ctDNA changes of KRAS G12C in a Phase 2 trial of adagrasib in patients with advanced, KRAS G12C-mutant lung cancer., Experimental Design: We performed serial droplet digital PCR (ddPCR) and plasma NGS on 60 KRAS G12C-mutant patients with lung cancer that participated in cohort A of the KRYSTAL-1 clinical trial. We analyzed the change in ctDNA at 2 specific intervals: Between cycles 1 and 2 and at cycle 4. Changes in ctDNA were compared with clinical and radiographic response., Results: We found that, in general, a maximal response in KRAS G12C ctDNA levels could be observed during the initial approximately 3-week treatment period, well before the first scan at approximately 6 weeks. 35 patients (89.7%) exhibited a decrease in KRAS G12C cfDNA >90% and 33 patients (84.6%) achieved complete clearance by cycle 2. Patients with complete ctDNA clearance at cycle 2 showed an improved objective response rate (ORR) compared with patients with incomplete ctDNA clearance (60.6% vs. 33.3%). Furthermore, complete ctDNA clearance at cycle 4 was associated with an improved overall survival (14.7 vs. 5.4 months) and progression-free survival (HR, 0.3)., Conclusions: These results support using early plasma response of KRAS G12C assessed at approximately 3 weeks to anticipate the likelihood of a favorable objective clinical response., (©2023 American Association for Cancer Research.)

With the combination of KRASG12C and EGFR inhibitors, KRAS is becoming a druggable target in colorectal cancer. However, secondary resistance limits its efficacy. Using cell lines, patient-derived xenografts, and patient samples, we detected a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drugs at progression. Serial analysis of patient blood samples on treatment demonstrates that most of these alterations are detected at a low frequency except for KRASG12C amplification, a recurrent resistance mechanism that rises in step with clinical progression. Upon drug withdrawal, resistant cells with KRASG12C amplification undergo oncogene-induced senescence, and progressing patients experience a rapid fall in levels of this alteration in circulating DNA. In this new state, drug resumption is ineffective as mTOR signaling is elevated. However, our work exposes a potential therapeutic vulnerability, whereby therapies that target the senescence response may overcome acquired resistance., Significance: Clinical resistance to KRASG12C-EGFR inhibition primarily prevents suppression of ERK signaling. Most resistance mechanisms are subclonal, whereas KRASG12C amplification rises over time to drive a higher portion of resistance. This recurrent resistance mechanism leads to oncogene-induced senescence upon drug withdrawal and creates a potential vulnerability to senolytic approaches. This article is highlighted in the In This Issue feature, p. 1., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background: Adagrasib, a KRAS G12C inhibitor, irreversibly and selectively binds KRAS G12C , locking it in its inactive state. Adagrasib showed clinical activity and had an acceptable adverse-event profile in the phase 1-1b part of the KRYSTAL-1 phase 1-2 study., Methods: In a registrational phase 2 cohort, we evaluated adagrasib (600 mg orally twice daily) in patients with KRAS G12C -mutated non-small-cell lung cancer (NSCLC) previously treated with platinum-based chemotherapy and anti-programmed death 1 or programmed death ligand 1 therapy. The primary end point was objective response assessed by blinded independent central review. Secondary end points included the duration of response, progression-free survival, overall survival, and safety., Results: As of October 15, 2021, a total of 116 patients with KRAS G12C -mutated NSCLC had been treated (median follow-up, 12.9 months); 98.3% had previously received both chemotherapy and immunotherapy. Of 112 patients with measurable disease at baseline, 48 (42.9%) had a confirmed objective response. The median duration of response was 8.5 months (95% confidence interval [CI], 6.2 to 13.8), and the median progression-free survival was 6.5 months (95% CI, 4.7 to 8.4). As of January 15, 2022 (median follow-up, 15.6 months), the median overall survival was 12.6 months (95% CI, 9.2 to 19.2). Among 33 patients with previously treated, stable central nervous system metastases, the intracranial confirmed objective response rate was 33.3% (95% CI, 18.0 to 51.8). Treatment-related adverse events occurred in 97.4% of the patients - grade 1 or 2 in 52.6% and grade 3 or higher in 44.8% (including two grade 5 events) - and resulted in drug discontinuation in 6.9% of patients., Conclusions: In patients with previously treated KRAS G12C -mutated NSCLC, adagrasib showed clinical efficacy without new safety signals. (Funded by Mirati Therapeutics; ClinicalTrials.gov number, NCT03785249.)., (Copyright © 2022 Massachusetts Medical Society.)

This study describes our efforts to further the field of noninvasive diagnostics, specifically in the area of liquid biopsies in oncology. We employed laser scanning cytometry using highly selective antibodies to interrogate circulating tumor cells (CTCs) that were isolated using ApoStream(®) technology to identify folate receptor alpha (FRα)-positive cells. We demonstrate that FRα(+) CTCs can be isolated from patients with metastatic cancers, including NSCLC adenocarcinoma, breast cancer, and ovarian cancer, whereas squamous cell lung cancer and normal healthy controls were devoid of FRα(+) CTCs. We believe that the developed methodology will have applications in both the diagnosis and the monitoring of FRα-expressing cancers. Folate receptor alpha (FRα) expression may have utility as a potential diagnostic and therapeutic target in solid tumors. As tissue samples are not always available for patient screening, this study evaluated a noninvasive assay in CTCs from blood samples to detect FRα expression. The presence of FRα(+) CTCs enriched using ApoStream(®) and detected using laser capture cytometry was evaluated in blood samples from cancer patients [NSCLC adenocarcinoma (n = 14), breast cancer (n = 20), ovarian cancer (n = 6), and squamous lung cancer patients (n = 6)] and healthy subjects (n = 20). The data demonstrated that FRα(+) CTCs were detected in blood from NSCLC adenocarcinoma, breast, and ovarian cancer patients, whereas squamous cell lung cancer patients and normal healthy controls lacked FRα(+) CTCs as previously known. We demonstrate that CTCs captured using ApoStream(®) can be used to detect FRα(+) CTCs and may have clinical utility as a real-time liquid biopsy for assessing FRα levels in cancer patients.

Background: The current study was conducted to evaluate the safety and biological activity of dual inhibition of the vascular endothelial growth factor (VEGF) pathway with combined bevacizumab and cediranib (a VEGF receptor tyrosine kinase inhibitor)., Methods: This was a 3 + 3 dose escalation study in patients with advanced solid tumors. Cediranib was given orally daily for 21 days and bevacizumab intravenously every 2 weeks. Pharmacokinetics and correlates (nitric oxide synthase, nitrate oxide, and circulating tumor cells) were assessed., Results: Fifty-one patients were treated. Dose-limiting toxicities (DLTs) (grade 3-4; graded according to the National Cancer Institute Common Terminology Criteria of Adverse Events [version 3.0]) observed included 1 patient with chest pain, 1 patient with fatigue, 2 patients with thrombocytopenia, 3 patients with hypertension (1 with intracranial hemorrhage), and 1 patient with grade 5 hemoptysis. Moreover, 2 patients presented with grade 3 intracranial bleeding beyond the DLT window. Dose level 2 (cediranib at a dose of 20 mg/day and bevacizumab at a dose of 5 mg/kg every 2 weeks) was selected as the recommended phase 2 dose (RP2D); 17 patients were treated at dose level 2 with 1 DLT and no intracranial bleeding or severe hypertension reported. Pharmacokinetics of cediranib at dose level 3 demonstrated a 46% to 77% increase in area under the curve (0-24 hours) on cycle 1 day 1 compared with historical controls. Four patients attained partial remissions: inflammatory breast cancer (-54%), basal cell carcinoma (-33%), alveolar soft part sarcoma (-33%), and synovial sarcoma (-32%). Patients with a lower circulating tumor cell count (< 30) at the predose period had a longer time to tumor progression (P = .024, log-rank test)., Conclusions: Cediranib at a dose of 20 mg/day and bevacizumab at a dose of 5 mg/kg every 2 weeks was found to be the RP2D. Activity in several tumor types was noted. Central nervous system bleeding and severe hypertension were observed at doses above the RP2D., (© 2014 American Cancer Society.)

Ser/Thr protein kinase CK2 regulates multiple processes that play important roles in the sensitivity of cancer to epidermal growth factor receptor targeting therapeutics, including PI3K-Akt-mTOR signaling, Hsp90 activity, and inhibition of apoptosis. We hypothesized that top-down inhibition of EGFR, combined with lateral suppression of multiple oncogenic pathways by targeting CK2, would create a pharmacologic synthetic lethal event and result in an improved cancer therapy compared to EGFR inhibition alone. This hypothesis was tested by combining CX-4945, a first-in-class clinical stage inhibitor of CK2, with the EGFR tyrosine kinase inhibitor, erlotinib, in vitro and in vivo in models of non-small cell lung carcinoma, NCI-H2170, and squamous cell carcinoma, A431. Our results demonstrate that combination of CX-4945 with erlotinib results in enhanced attenuation of the PI3K-Akt-mTOR pathway. We also observed an increase in apoptosis, synergistic killing of cancer cells in vitro, as well as improved antitumor efficacy in vivo. Taken together, these data position CK2 as a valid pharmacologic target for drug combinations and support further evaluation of CX-4945 in combination with EGFR targeting agents., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Increased transcription of ribosomal RNA genes (rDNA) by RNA Polymerase I is a common feature of human cancer, but whether it is required for the malignant phenotype remains unclear. We show that rDNA transcription can be therapeutically targeted with the small molecule CX-5461 to selectively kill B-lymphoma cells in vivo while maintaining a viable wild-type B cell population. The therapeutic effect is a consequence of nucleolar disruption and activation of p53-dependent apoptotic signaling. Human leukemia and lymphoma cell lines also show high sensitivity to inhibition of rDNA transcription that is dependent on p53 mutational status. These results identify selective inhibition of rDNA transcription as a therapeutic strategy for the cancer specific activation of p53 and treatment of hematologic malignancies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Accelerated proliferation of solid tumor and hematologic cancer cells is linked to accelerated transcription of rDNA by the RNA polymerase I (Pol I) enzyme to produce elevated levels of rRNA (rRNA). Indeed, upregulation of Pol I, frequently caused by mutational alterations among tumor suppressors and oncogenes, is required for maintenance of the cancer phenotype and forms the basis for seeking selective inhibitors of Pol I as anticancer therapeutics. 2-(4-Methyl-[1,4]diazepan-1-yl)-5-oxo-5H-7-thia-1,11b-diaza-benzo[c]fluorene-6-carboxylic acid (5-methyl-pyrazin-2-ylmethyl)-amide (CX-5461, 7c) has been identified as the first potent, selective, and orally bioavailable inhibitor of RNA Pol I transcription with in vivo activity in tumor growth efficacy models. The preclinical data support the development of CX-5461 as an anticancer drug with potential for activity in several types of cancer.

Drug combination therapies are commonly used for the treatment of cancers to increase therapeutic efficacy, reduce toxicity, and decrease the incidence of drug resistance. Although drug combination therapies were originally devised primarily by empirical methods, the increased understanding of drug mechanisms and the pathways they modulate provides a unique opportunity to design combinations that are based on mechanistic rationale. We have identified protein kinase CK2 as a promising therapeutic target for combination therapy, because CK2 regulates not just one but many oncogenic pathways and processes that play important roles in drug resistance, including DNA repair, epidermal growth factor receptor signaling, PI3K/AKT/mTOR signaling, Hsp90 machinery activity, hypoxia, and interleukin-6 expression. In this article, we show that CX-4945, a clinical stage selective small molecule inhibitor of CK2, blocks the DNA repair response induced by gemcitabine and cisplatin and synergizes with these agents in models of ovarian cancer. Mechanistic studies show that the enhanced activity is a result of inactivation of XRCC1 and MDC1, two mediator/adaptor proteins that are essential for DNA repair and that require phosphorylation by CK2 for their function. These data position CK2 as a valid pharmacologic target for intelligent drug combinations and support the evaluation of CX-4945 in combination with gemcitabine and platinum-based chemotherapeutics in the clinical setting., (©2012 AACR.)

Structure-activity relationship analysis in a series of 3-(5-((2-oxoindolin-3-ylidene)methyl)furan-2-yl)amides identified compound 13, a pan-Pim kinases inhibitor with excellent biochemical potency and kinase selectivity. Compound 13 exhibited in vitro synergy with chemotherapeutics and robust in vivo efficacy in two Pim kinases driven tumor models.

A novel class of pan-Pim kinase inhibitors was designed by modifying the CK2 inhibitor CX-4945. Introduction of a triazole or secondary amide functionality on the C-7 position and 2'-halogenoanilines on C-5 resulted in potent inhibitors of the Pim-1 and Pim-2 isoforms, with many analogs active at single digit nanomolar concentrations. The molecules inhibited the phosphorylation at Serine 112 of the apoptosis effector BAD, and had potent antiproliferative effects on the AML cell line MV-4-11 (IC(50) <30 nM). This work delivers an excellent lead-optimization platform for Pim targeting anticancer therapies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Inflammatory breast cancer is driven by pro-angiogenic and pro-inflammatory cytokines. One of them Interleukin-6 (IL-6) is implicated in cancer cell proliferation and survival, and promotes angiogenesis, inflammation and metastasis. While IL-6 has been shown to be upregulated by several oncogenes, the mechanism behind this phenomenon is not well characterized. Here we demonstrate that the pleotropic Serine/Threonine kinase CK2 is implicated in the regulation of IL-6 expression in a model of inflammatory breast cancer. We used siRNAs targeted toward CK2 and a selective small molecule inhibitor of CK2, CX-4945, to inhibit the expression and thus suppress the secretion of IL-6 in in vitro as well as in vivo models. Moreover, we report that in a clinical trial, CX-4945 was able to dramatically reduce IL-6 levels in plasma of an inflammatory breast cancer patient. Our data shed a new light on the regulation of IL-6 expression and position CX-4945 and potentially other inhibitors of CK2, for the treatment of IL-6-driven cancers and possibly other diseases where IL-6 is instrumental, including rheumatoid arthritis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

5-(3-Chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first clinical stage inhibitor of protein kinase CK2 for the treatment of cancer, is representative of a new class of CK2 inhibitors with K(i) values in the low nanomolar range and unprecedented selectivity versus other kinases. Here we present the crystal structure of the complexes of CX-4945 and two analogues (CX-5011 and CX-5279) with the catalytic subunit of human CK2. Consistent with their ATP-competitive mode of inhibition, all three compounds bind in the active site of CK2 (type I inhibitors). The tricyclic scaffold of the inhibitors superposes on the adenine of ATP, establishing multiple hydrophobic interactions with the binding cavity. The more extended scaffold, as compared to that of ATP, allows the carboxylic function, shared by all three ligands, to penetrate into the deepest part of the active site where it makes interactions with conserved water W1 and Lys-68, thus accounting for the crucial role of this negatively charged group in conferring high potency to this class of inhibitors. The presence of a pyrimidine in CX-5011 and in CX-5279 instead of a pyridine (as in CX-4945) ring is likely to account for the higher specificity of these compounds whose Gini coefficients, calculated by profiling them against panels of 102 and/or 235 kinases, are significantly higher than that of CX-4945 (0.735 and 0.755, respectively, vs 0.615), marking the highest selectivity ever reported for CK2 inhibitors.

In this article we describe the preclinical characterization of 5-(3-chlorophenylamino) benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first orally available small molecule inhibitor of protein CK2 in clinical trials for cancer. CX-4945 was optimized as an ATP-competitive inhibitor of the CK2 holoenzyme (Ki = 0.38 nM). Iterative synthesis and screening of analogs, guided by molecular modeling, led to the discovery of orally available CX-4945. CK2 promotes signaling in the Akt pathway and CX-4945 suppresses the phosphorylation of Akt as well as other key downstream mediators of the pathway such as p21. CX-4945 induced apoptosis and caused cell cycle arrest in cancer cells in vitro. CX-4945 exhibited a dose-dependent antitumor activity in a xenograft model of PC3 prostate cancer model and was well tolerated. In vivo time-dependent reduction in the phosphorylation of the biomarker p21 at T145 was observed by immunohistochemistry. Inhibition of the newly validated CK2 target by CX-4945 represents a fresh therapeutic strategy for cancer.

We describe the discovery of novel potent substituted pyrimido[4,5-c]quinoline ATP-competitive inhibitors of protein kinase CK2. A binding model of the inhibitors with the protein was elaborated on the basis of SAR and revealed various modes of interaction with the hinge region. Representative analog 14k (CK2 IC(50)=9 nM) showed anti-viral activity at nanomolar concentrations against HIV-1. Orally available compound 7e (CK2 IC(50)=3 nM) reduced pain in the phase II of a murine formalin model. These preliminary data confirm that properly optimized CK2 inhibitors may be used for anti-viral and pain therapy., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Deregulated ribosomal RNA synthesis is associated with uncontrolled cancer cell proliferation. RNA polymerase (Pol) I, the multiprotein complex that synthesizes rRNA, is activated widely in cancer. Thus, selective inhibitors of Pol I may offer a general therapeutic strategy to block cancer cell proliferation. Coupling medicinal chemistry efforts to tandem cell- and molecular-based screening led to the design of CX-5461, a potent small-molecule inhibitor of rRNA synthesis in cancer cells. CX-5461 selectively inhibits Pol I-driven transcription relative to Pol II-driven transcription, DNA replication, and protein translation. Molecular studies demonstrate that CX-5461 inhibits the initiation stage of rRNA synthesis and induces both senescence and autophagy, but not apoptosis, through a p53-independent process in solid tumor cell lines. CX-5461 is orally bioavailable and demonstrates in vivo antitumor activity against human solid tumors in murine xenograft models. Our findings position CX-5461 for investigational clinical trials as a potent, selective, and orally administered agent for cancer treatment., (©2010 AACR.)

Herein we chronicle the discovery of CX-4945 (25n), a first-in-class, orally bioavailable ATP-competitive inhibitor of protein kinase CK2 in clinical trials for cancer. CK2 has long been considered a prime cancer drug target because of the roles of deregulated and overexpressed CK2 in cancer-promoting prosurvival and antiapoptotic pathways. These biological properties as well as the suitability of CK2's small ATP binding site for the design of selective inhibitors, led us to fashion novel therapeutic agents for cancer. The optimization leading to 25n (K(i) = 0.38 nM) was guided by molecular modeling, suggesting a strong binding of 25n resulting from a combination of hydrophobic interactions, an ionic bridge with Lys68, and hydrogen bonding with the hinge region. 25n was found to be highly selective, orally bioavailable across species (20-51%) and efficacious in xenograft models. The discovery of 25n will allow the therapeutic targeting of CK2 in humans for the first time.

Malignant transformation and maintenance of the malignant phenotype depends on oncogenic and non-oncogenic proteins that are essential to mediate oncogene signaling and to support the altered physiologic demands induced by transformation. Protein kinase CK2 supports key prosurvival signaling pathways and represents a prototypical non-oncogene. In this study, we describe CX-4945, a potent and selective orally bioavailable small molecule inhibitor of CK2. The antiproliferative activity of CX-4945 against cancer cells correlated with expression levels of the CK2α catalytic subunit. Attenuation of PI3K/Akt signaling by CX-4945 was evidenced by dephosphorylation of Akt on the CK2-specific S129 site and the canonical S473 and T308 regulatory sites. CX-4945 caused cell-cycle arrest and selectively induced apoptosis in cancer cells relative to normal cells. In models of angiogenesis, CX-4945 inhibited human umbilical vein endothelial cell migration, tube formation, and blocked CK2-dependent hypoxia-induced factor 1 alpha (HIF-1α) transcription in cancer cells. When administered orally in murine xenograft models, CX-4945 was well tolerated and demonstrated robust antitumor activity with concomitant reductions of the mechanism-based biomarker phospho-p21 (T145). The observed antiproliferative and anti-angiogenic responses to CX-4945 in tumor cells and endothelial cells collectively illustrate that this compound exerts its antitumor effects through inhibition of CK2-dependent signaling in multiple pathways. Finally, CX-4945 is the first orally bioavailable small molecule inhibitor of CK2 to advance into human clinical trials, thereby paving the way for an entirely new class of targeted treatment for cancer., (©2010 AACR.)

Purpose: P-cadherin is a membrane glycoprotein that functionally mediates tumor cell adhesion, proliferation, and invasiveness. We characterized the biological properties of PF-03732010, a human monoclonal antibody against P-cadherin, in cell-based assays and tumor models., Experimental Design: The affinity, selectivity, and cellular inhibitory activity of PF-03732010 were tested in vitro. Multiple orthotopic and metastatic tumor models were used for assessing the antitumor and antimetastatic activities of PF-03732010. Treatment-associated pharmacodynamic changes were also investigated., Results: PF-03732010 selectively inhibits P-cadherin-mediated cell adhesion and aggregation in vitro. In the P-cadherin-overexpressing tumor models, including MDA-MB-231-CDH3, 4T1-CDH3, MDA-MB-435HAL-CDH3, HCT116, H1650, PC3M-CDH3, and DU145, PF-03732010 inhibited the growth of primary tumors and metastatic progression, as determined by bioluminescence imaging. Computed tomography imaging, H&E stain, and quantitative PCR analysis confirmed the antimetastatic activity of PF-03732010. In contrast, PF-03732010 did not show antitumor and antimetastatic efficacy in the counterpart tumor models exhibiting low P-cadherin expression. Mechanistic studies via immunofluorescence, immunohistochemical analyses, and 3'-[(18)F]fluoro-3'-deoxythymidine-positron emission tomography imaging revealed that PF-03732010 suppressed P-cadherin levels, caused degradation of membrane β-catenin, and concurrently suppressed cytoplasmic vimentin, resulting in diminished metastatic capacity. Changes in the levels of Ki67, caspase-3, and 3'-[(18)F]fluoro-3'-deoxythymidine tracer uptake also indicated antiproliferative activity and increased apoptosis in the tested xenografts., Conclusions: These findings suggest that interrupting the P-cadherin signaling pathway may be a novel therapeutic approach for cancer therapy. PF-03732010 is presently undergoing evaluation in Phase 1 clinical trials., (©2010 AACR.)

Hallmark deregulated signaling in cancer cells drives excessive ribosome biogenesis within the nucleolus, which elicits unbridled cell growth and proliferation. The rate-limiting step of ribosome biogenesis is synthesis of rRNA (building blocks of ribosomes) by RNA Polymerase I (Pol I). Numerous kinase pathways and products of proto-oncogenes can up-regulate Pol I, whereas tumor suppressor proteins can inhibit rRNA synthesis. In tumorigenesis, activating mutations in certain cancer-associated kinases and loss-of-function mutations in tumor suppressors lead to deregulated signaling that stimulates Pol I transcription with resultant increases in ribosome biogenesis, protein synthesis, cell growth, and proliferation. Certain anticancer therapeutics, such as cisplatin and 5-fluorouracil, reportedly exert, at least partially, their activity through disruption of ribosome biogenesis, yet many prime targets for anticancer drugs within the ribosome synthetic machinery of the nucleolus remain largely unexploited. Herein, we describe CX-3543, a small molecule nucleolus-targeting agent that selectively disrupts nucleolin/rDNA G-quadruplex complexes in the nucleolus, thereby inhibiting Pol I transcription and inducing apoptosis in cancer cells. CX-3543 is the first G-quadruplex interactive agent to enter human clinical trials, and it is currently under evaluation against carcinoid/neuroendocrine tumors in a phase II clinical trial.

Purpose: Checkpoint kinase 1 (Chk1) plays a critical role in the activation of mitotic spindle checkpoint and DNA damage checkpoint. We examined the preclinical use of the Chk1 inhibitor PF-00477736 as a docetaxel-sensitizing agent. Specifically, we investigated the correlation between PF-00477736-mediated modulation of biomarkers and the sensitization of docetaxel efficacy., Experimental Design: In vitro and in vivo studies using COLO205 and other cell lines were done to assess PF-00477736-induced enhancement of docetaxel efficacy and effects on associated biomarkers., Results: PF-00477736 significantly enhanced the docetaxel-induced efficacy in tumor cells and xenografts. Docetaxel induced dose- and time-dependent increase in the levels of phosphorylated Chk1 (Ser(345)), phosphorylated histone H3 (Ser(10)), and gammaH2AX foci and promoted the cytoplasmic localization of phosphorylated Cdc25C (Ser(216)). PF-00477736 cotreatment suppressed docetaxel-induced changes in phosphorylated histone H3 and cytoplasmic phosphorylated Cdc25C (Ser(216)) levels and concurrently sensitized the docetaxel-induced apoptosis. Docetaxel alone or in combination with PF-00477736 induced significant antiproliferative activity in xenografts, shown via [18F]FLT-PET imaging. However, changes in [18F]FLT uptake did not reflect the potentiation of docetaxel efficacy. In contrast, bioluminescence imaging showed that PF-00477736 sensitized docetaxel-induced suppression of tumor survival., Conclusions: Docetaxel triggers mitotic spindle checkpoint activation at low concentrations and activates both the DNA damage checkpoint and the spindle checkpoint at high concentrations. In combination with docetaxel, PF-00477736 abrogates the mitotic checkpoint, as well as the DNA damage checkpoint, and results in sensitization to docetaxel. Chk1 inhibitor PF-00477736 offers a therapeutic potential for the enhancement of taxane therapy.

New approaches to prevent and reverse UV damage are needed to combat rising sunlight-induced skin cancer rates. Mouse studies have shown that oral or topical caffeine promotes elimination of UV-damaged keratinocytes through apoptosis and markedly inhibits subsequent skin cancer development. This potentially important therapeutic effect has not been studied in human skin cells. Here, we use primary human keratinocytes to examine which of several caffeine effects mediates this process. In these cells, caffeine more than doubled apoptosis after 75 mJ cm(-2) of ultraviolet light B (UVB). Selectively targeting two of caffeine's known effects did not alter UVB-induced apoptosis: inhibition of ataxia-telangiectasia mutated and augmentation of cyclic AMP levels. In contrast, siRNA against ataxia-telangiectasia and Rad3-related (ATR) doubled apoptosis after UV through a p53-independent mechanism. Caffeine did not further augment apoptosis after UVB in cells in which ATR had been specifically depleted, suggesting that a key target of caffeine in this effect is ATR. Inhibition of a central ATR target, checkpoint kinase 1 (Chk1), through siRNA or a new and highly specific inhibitor (PF610666) also augmented UVB-induced apoptosis. These data suggest that a relevant target of caffeine is the ATR-Chk1 pathway and that inhibiting ATR or Chk1 might have promise in preventing or reversing UV damage.

Purpose: Tumors grafted s.c. or under the mammary fat pad (MFP) rarely develop efficient metastasis. By applying bioluminescence imaging (BLI) technology, the MDA-MB-435-HAL-Luc subrenal capsule (SRC) model was compared with the MFP model for disease progression, metastatic potential, and response to therapy., Experimental Design: The luciferase-expressing MDA-MB-435-HAL-Luc cell line was used in both MFP and SRC models. BLI technology allowed longitudinal assessment of disease progression and the therapeutic response to PD-0332991, Avastin, and docetaxel. Immunohistochemical analysis of Ki67 and CD31 staining in the primary tumors was compared in these models. Caliper measurement was used in the MFP model to validate the BLI quantification of primary tumors., Results: The primary tumors in MDA-MB-435-HAL-Luc MFP and SRC models displayed comparable growth rates and vascularity. However, tumor-bearing mice in the SRC model developed lung metastases much earlier (4 weeks) than in the MFP model (>7 weeks), and the metastatic progression contributed significantly to the survival time. In the MFP model, BLI and caliper measurements were comparable for quantifying palpable tumors, but BLI offered an advantage for detecting the primary tumors that fell below a palpable threshold and for visualizing metastases. In the SRC model, BLI allowed longitudinal assessment of the antitumor and antimetastatic effects of PD-0332991, Avastin, and docetaxel, and the results correlated with the survival benefits of these agents., Conclusions: The MDA-MB-435-HAL-Luc SRC model and the MFP model displayed differences in disease progression. BLI is an innovative approach for developing animal models and creates opportunities for improving preclinical evaluations of anticancer agents.

Checkpoints are present in all phases of the cell cycle and are regarded as the gatekeepers maintaining the integrity of the genome. Many conventional agents used to treat cancer impart damage to the genome and activate cell cycle checkpoints. Many tumors are defective in the tumor suppressor p53 and therefore lack a functional G(1) checkpoint. In these tumors, however, the S-G(2) checkpoints remain intact and, in response to DNA damage, arrest cell cycle progression allowing time for DNA repair. Checkpoint kinase 1 (Chk1) is a key element in the DNA damage response pathway and plays a crucial role in the S-G(2)-phase checkpoints. Inhibiting Chk1 represents a therapeutic strategy for creating a "synthetic lethal" response by overriding the last checkpoint defense of tumor cells against the lethal damage induced by DNA-directed chemotherapeutic agents. Chk1 inhibition is consistent with emerging targeted therapies aiming to exploit molecular differences between normal and cancer cells. Adding a Chk1 inhibitor to DNA-damaging cytotoxic therapy selectively targets tumors with intrinsic checkpoint defects while minimizing toxicity in checkpoint-competent normal cells. PF-00477736 was identified as a potent, selective ATP-competitive small-molecule inhibitor that inhibits Chk1 with a K(i) of 0.49 nM. PF-00477736 abrogates cell cycle arrest induced by DNA damage and enhances cytotoxicity of clinically important chemotherapeutic agents, including gemcitabine and carboplatin. In xenografts, PF-00477736 enhanced the antitumor activity of gemcitabine in a dose-dependent manner. PF-00477736 combinations were well tolerated with no exacerbation of side effects commonly associated with cytotoxic agents.

The cocrystal structure of a library hit was used to design a novel series of CHK1 inhibitors. The new series retained the critical hydrogen-bonding groups of the resorcinol moiety for binding but lacked the phenolic anilide moiety. The newly designed compounds exhibited similar enzymatic activity, while demonstrating increased cellular potency. Compound 10c, showing no single agent effect, potentiated the antiproliferative effect of Gemcitabine in both prostate and breast cancer cell lines.

Highly potent human glucagon receptor (hGluR) antagonists have been prepared employing both medicinal chemistry and targeted libraries based on modification of the core (proximal) dimethoxyphenyl group, the benzyl ether linkage, as well as the (distal) benzylic aryl group of the lead 2, 3-cyano-4-hydroxybenzoic acid (3,5-dimethoxy-4-isopropylbenzyloxybenzylidene)hydrazide. Electron-rich proximal aryl moieties such as mono- and dimethoxy benzenes, naphthalenes, and indoles were found to be active. The SAR was found to be quite insensitive regarding the linkage to the distal aryl group, since long and short as well as polar and apolar linkers gave highly potent compounds. The presence of a distal aryl group was not crucial for obtaining high binding affinity to the hGluR. In many cases, however, the affinity could be further optimized with substituted distal aryl groups. Representative compounds have been tested for in vitro metabolism, and structure-metabolism relationships are described. These efforts lead to the discovery of 74, NNC 25-2504, 3-cyano-4-hydroxybenzoic acid [1-(2,3,5,6-tetramethylbenzyl)-1H-indol-4-ylmethylene]hydrazide, with low in vitro metabolic turnover. 74 was a highly potent noncompetitive antagonist of the human glucagon receptor (IC(50) = 2.3 nM, K(B) = 760 pM) and of the isolated rat receptor (IC(50) = 430 pM, K(B) = 380 pM). Glucagon-stimulated glucose production from isolated primary rat hepatocytes was inhibited competitively by 74 (K(i) = 14 nM). This compound was orally available in dogs (F(po) = 15%) and was active in a glucagon-challenged rat model of hyperglucagonemia and hyperglycemia.

A series of alkylidene hydrazide derivatives containing an alkoxyaryl moiety was optimized. The resulting hydrazide-ethers were competitive antagonists at the human glucagon receptor. Pharmacokinetic experiments showed fast clearance of most of the compounds tested. A representative compound [4-hydroxy-3-cyanobenzoic acid (4-isopropylbenzyloxy-3,5-dimethoxymethylene)hydrazide] with an IC50 value of 20 nM was shown to reduce blood glucose levels in fasted rats.

High throughput screening of our small molecule combinatorial library identified a class of benzoylnaphthalenehydrazones with modest affinity for the human glucagon receptor. Optimization of this initial hit through a series of targeted libraries and traditional medicinal chemistry led to ligands with nanomolar affinities. Pharmacological evaluation demonstrated that these ligands were competitive glucagon receptor antagonists. Intravenous administration of a representative benzoylnaphthalenehydrazone into rats attenuated glucagon-stimulated glucose levels.