Computational Modeling of Drug Response Identifies Mutant-Specific Constraints for Dosing panRAF and MEK Inhibitors in Melanoma.

Purpose: This study explores the potential of pre-clinical in vitro cell line response data and computational modeling in identifying the optimal dosage requirements of pan-RAF (Belvarafenib) and MEK (Cobimetinib) inhibitors in melanoma treatment. Our research is motivated by the critical role of drug combinations in enhancing anti-cancer responses and the need to close the knowledge gap around selecting effective dosing strategies to maximize their potential.

Results: In a drug combination screen of 43 melanoma cell lines, we identified specific dosage landscapes of panRAF and MEK inhibitors for NRAS vs.

Computational modeling of drug response identifies mutant-specific constraints for dosing panRAF and MEK inhibitors in melanoma.

Purpose: This study explores the potential of preclinical cell line response data and computational modeling in identifying optimal dosage requirements of pan-RAF (Belvarafenib) and MEK (Cobimetinib) inhibitors in melanoma treatment. Our research is motivated by the critical role of drug combinations in enhancing anti-cancer responses and the need to close the knowledge gap around selecting effective dosing strategies to maximize their potential.

Results: In a drug combination screen of 43 melanoma cell lines, we identified unique dosage landscapes of panRAF and MEK inhibitors for NRAS vs BRAF mutant melanomas.

Allosteric Inhibitors of the SARS-COV-2 Papain-like Protease Domain Induce Proteasomal Degradation of Its Parent Protein NSP3.

The papain-like protease of SARS-COV-2 is essential for viral replication and pathogenesis. Its location within a much larger multifunctional protein, NSP3, makes it an ideal candidate for a targeted degradation approach capable of eliminating multiple functions with a single-molecule treatment. In this work, we have developed a HiBiT-based cellular model to study NSP3 degradation and used this platform for the discovery of monovalent NSP3 degraders.

Ternary complex dissociation kinetics contribute to mutant-selective EGFR degradation.

Targeted degradation of proteins by chimeric heterobifunctional degraders has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a degrader remain poorly understood, and potent target engagement by a degrader does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an epidermal growth factor receptor (EGFR) degrader that potently binds both wild-type and mutant EGFR, but only degrades EGFR mutant variants.

CRAF dimerization with ARAF regulates KRAS-driven tumor growth.

KRAS, which is mutated in ∼30% of all cancers, activates the RAF-MEK-ERK signaling cascade. CRAF is required for growth of KRAS mutant lung tumors, but the requirement for CRAF kinase activity is unknown. Here, we show that subsets of KRAS mutant tumors are dependent on CRAF for growth.

Identifying transcriptional programs underlying cancer drug response with TraCe-seq.

Genetic and non-genetic heterogeneity within cancer cell populations represent major challenges to anticancer therapies. We currently lack robust methods to determine how preexisting and adaptive features affect cellular responses to therapies. Here, by conducting clonal fitness mapping and transcriptional characterization using expressed barcodes and single-cell RNA sequencing (scRNA-seq), we have developed tracking differential clonal response by scRNA-seq (TraCe-seq).

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a Pyridopyridazinone pan-RAF Kinase Inhibitor.

Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (), which features a unique pyrido[2,3-]pyridazin-8(7)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations.

ARAF mutations confer resistance to the RAF inhibitor belvarafenib in melanoma.

Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAF-mutant melanoma, they are ineffective in non-BRAF mutant cells. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAF- and NRAS-mutant melanomas.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a 5-Fluoro-4-(3)-quinazolinone Aryl Urea pan-RAF Kinase Inhibitor.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule.

Pharmacological Induction of RAS-GTP Confers RAF Inhibitor Sensitivity in KRAS Mutant Tumors.

Targeting KRAS mutant tumors through inhibition of individual downstream pathways has had limited clinical success. Here we report that RAF inhibitors exhibit little efficacy in KRAS mutant tumors. In combination drug screens, MEK and PI3K inhibitors synergized with pan-RAF inhibitors through an RAS-GTP-dependent mechanism.

Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening.

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines.

Phase I dose-escalation trial of checkpoint kinase 1 inhibitor MK-8776 as monotherapy and in combination with gemcitabine in patients with advanced solid tumors.

Purpose: We determined the safety, pharmacokinetics, pharmacodynamics, and recommended phase II dose of MK-8776 (SCH 900776), a potent, selective checkpoint kinase 1 (Chk1) inhibitor, as monotherapy and in combination with gemcitabine in a first-in-human phase I clinical trial in patients with advanced solid tumor malignancies.

Patients And Methods: Forty-three patients were treated by intravenous infusion with MK-8776 at seven dose levels ranging from 10 to 150 mg/m(2) as monotherapy and then in combination with gemcitabine 800 mg/m(2) (part A, n = 26) or gemcitabine 1,000 mg/m(2) (part B, n = 17). Forty percent of patients had three or more prior treatment regimens, and one third of patients had previously received gemcitabine.

A functional approach reveals a genetic and physical interaction between ribonucleotide reductase and CHK1 in mammalian cells.

Ribonucleotide reductase (RNR) enzyme is composed of the homodimeric RRM1 and RRM2 subunits, which together form a heterotetramic active enzyme that catalyzes the de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. In this study, we show that ablation of RRM1 and RRM2 by siRNA induces G1/S phase arrest, phosphorylation of Chk1 on Ser345 and phosphorylation of γ-H2AX on S139. Combinatorial ablation of RRM1 or RRM2 and Chk1 causes a dramatic accumulation of γ-H2AX, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage.

The identification of novel 5'-amino gemcitabine analogs as potent RRM1 inhibitors.

The ribonucleotide reductase (RNR) enzyme is a heteromer of RRM1 and RRM2 subunits. The active enzyme catalyzes de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. Complexity in the generation of physiologically relevant, active RRM1/RRM2 heterodimers was perceived as limiting to the identification of selective RRM1 inhibitors by high-throughput screening of compound libraries and led us to seek alternative methods to identify lead series.

Targeting the replication checkpoint using SCH 900776, a potent and functionally selective CHK1 inhibitor identified via high content screening.

Checkpoint kinase 1 (CHK1) is an essential serine/threonine kinase that responds to DNA damage and stalled DNA replication. CHK1 is essential for maintenance of replication fork viability during exposure to DNA antimetabolites. In human tumor cell lines, ablation of CHK1 function during antimetabolite exposure led to accumulation of double-strand DNA breaks and cell death.

Phenotypic enhancement of thymidylate synthetase pathway inhibitors following ablation of Neil1 DNA glycosylase/lyase.

Inhibition of thymidine biosynthesis is a clinically-validated therapeutic approach for multiple cancers. Inhibition of thymidylate synthetase (TS) leads to a decrease in cellular TTP levels, replication stress and increased genomic incorporation of uridine (dUMP). Thus, inhibitors of this pathway (such as methotrexate) can drive a multitude of downstream cell cycle checkpoint and DNA repair processes.

Discovery of Dinaciclib (SCH 727965): A Potent and Selective Inhibitor of Cyclin-Dependent Kinases.

Inhibition of cyclin-dependent kinases (CDKs) has emerged as an attractive strategy for the development of novel oncology therapeutics. Herein is described the utilization of an in vivo screening approach with integrated efficacy and tolerability parameters to identify candidate CDK inhibitors with a suitable balance of activity and tolerability. This approach has resulted in the identification of SCH 727965, a potent and selective CDK inhibitor that is currently undergoing clinical evaluation.

Dinaciclib (SCH 727965), a novel and potent cyclin-dependent kinase inhibitor.

Cyclin-dependent kinases (CDK) are key positive regulators of cell cycle progression and attractive targets in oncology. SCH 727965 inhibits CDK2, CDK5, CDK1, and CDK9 activity in vitro with IC(50) values of 1, 1, 3, and 4 nmol/L, respectively. SCH 727965 was selected as a clinical candidate using a functional screen in vivo that integrated both efficacy and safety parameters.

Replication stress activates DNA polymerase alpha-associated Chk1.

Chk1 contributes to both intra-S and DNA damage checkpoint responses. Here, we show that depletion of DNA Polalpha and not Polepsilon or Poldelta by siRNA induces phosphorylation of Chk1 on Ser345, thus phenocopying antimetabolite exposure. Combinatorial ablation of DNA Polalpha and Chk1 causes an accumulation of gamma-H2A.

Versatile templates for the development of novel kinase inhibitors: Discovery of novel CDK inhibitors.

A series of four bicyclic cores were prepared and evaluated as cyclin-dependent kinase-2 (CDK2) inhibitors. From the in-vitro and cell-based analysis, the pyrazolo[1,5-a]pyrimidine core (represented by 9) emerged as the superior core for further elaboration in the identification of novel CDK2 inhibitors.

Role for BRG1 in cell cycle control and tumor suppression.

Human BRG1, a subunit of the Swi/Snf chromatin remodeling apparatus, has been implicated in regulation of cellular proliferation and is a candidate tumor suppressor. Reintroduction of BRG1 into a breast tumor cell line, ALAB, carrying a defined mutation in the BRG1 gene, induced growth arrest. Gene expression data revealed that the arrest may in part be accounted for by down-regulation of select E2F target genes such as cyclin E, but more dramatically, by up-regulation of mRNAs for the cyclin-dependent kinase inhibitors p21 and p15.