Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity.

Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl-/- mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.

Primary results and characterization of patients with exceptional outcomes in a phase 1b study combining PARP and MEK inhibition, with or without anti-PD-L1, for BRCA wild-type, platinum-sensitive, recurrent ovarian cancer.

BACKGROUND: This phase 1b study (ClinicalTrials.gov identifier NCT03695380) evaluated regimens combining PARP and MEK inhibition, with or without PD-L1 inhibition, for BRCA wild-type, platinum-sensitive, recurrent ovarian cancer (PSROC). METHODS: Patients with PSROC who had received one or two prior treatment lines were treated with 28-day cycles of cobimetinib 60 mg daily (days 1-21) plus niraparib 200 mg daily (days 1-28) with or without atezolizumab 840 mg (days 1 and 15). Stage 1 assessed safety before expansion to stage 2, which randomized patients who had BRCA wild-type PSROC to receive either doublet or triplet therapy, stratified by genome-wide loss of heterozygosity status (<16% vs. ≥16%; FoundationOne CDx assay) and platinum-free interval (≥6 to <12 vs. ≥12 months). Coprimary end points were safety and the investigator-determined objective response rate (ORR) according to Response Evaluation Criteria in Solid Tumors (RECIST). Potential associations between genetic parameters and efficacy were explored, and biomarker profiles of super-responders (complete response or those with progression-free survival [PFS] >15 months) and progressors (disease progression as the best response) were characterized. RESULTS: The ORR in patients who had BRCA wild-type PSROC was 35% (95% confidence interval, 20%-53%) with the doublet regimen (n = 37) and 27% (95% confidence interval, 14%-44%) with the triplet regimen (n = 37), and the median PFS was 6.0 and 7.4 months, respectively. Post-hoc analyses indicated more favorable ORR and PFS in the homologous recombination-deficiency-signature (HRDsig)-positive subgroup than in the HRDsig-negative subgroup. Tolerability was consistent with the known profiles of individual agents. NF1 and MKNK1 mutations were associated with sustained benefit from the doublet and triplet regimens, respectively. CONCLUSIONS: Chemotherapy-free doublet and triplet therapy demonstrated encouraging activity, including among patients who had BRCA wild-type, HRDsig-positive or HRDsig-negative PSROC harboring NF1 or MKNK1 mutations.

Quantifying KRAS G12C Covalent Drug Inhibitor Activity in Mouse Tumors Using Mass Spectrometry.

The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug activity in vivo for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry. We introduce a robust assay with an average intra-assay coefficient of variation (CV) of 4% and an interassay CV of 6% obtained from seven studies, enabling us to understand the relationship between KRAS G12C target occupancy and the therapeutic PD effect from mouse tumor samples. Further, the data demonstrated that the drug candidate GDC-6036, a KRAS G12C covalent inhibitor, shows dose-dependent target inhibition (KRAS G12C alkylation) and MAPK pathway inhibition, which correlate with high antitumor potency in the MIA PaCa-2 pancreatic xenograft model.

Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC-MS/MS Approach.

KRAS is one of the most frequently mutated oncogenes, with KRAS G12C recently becoming an actionable target for small molecule intervention. GDC-6036 is an investigational KRAS G12C inhibitor that acts by irreversibly binding to the switch II pocket of KRAS G12C when in the inactive GDP-bound state, thereby blocking GTP binding and activation. Assessing target engagement is an essential component of clinical drug development, helping to demonstrate mechanistic activity, guide dose selection, understand pharmacodynamics as it relates to clinical response, and explore resistance. Here, we report the development of an ultra-sensitive approach for assessing KRAS G12C engagement. Immunoaffinity enrichment with a commercially available anti-RAS antibody was combined with a targeted 2D-LC-MS/MS technique to quantify both free and GDC-6036-bound KRAS G12C proteins. A KRAS G12C-positive non-small cell lung cancer xenograft model was dosed with GDC-6036 to assess the feasibility of this assay for analyzing small core needle biopsies. As predicted, dose-dependent KRAS G12C engagement was observed. To date, a sensitivity of 0.08 fmol/µg of total protein has been achieved for both free and GDC-6036-bound KRAS G12C with as little as 4 µg of total protein extracted from human tumor samples. This sub-fmol/µg level of sensitivity provides a powerful potential approach to assess covalent inhibitor target engagement at the site of action using core needle tumor biopsies from clinical studies.

Disruption of IRE1α through its kinase domain attenuates multiple myeloma.

Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.

Detection and analysis of autophagy in the American alligator (Alligator mississippiensis).

In response to environmental temperature depression in the fall and winter, American alligators (Alligator mississippiensis) brumate. Brumation is characterized by lethargy, fasting, decreased metabolism, and decreased body temperature. During brumation, alligators will periodically emerge for basking or other encounters when environmental conditions permit. This sporadic activity and lack of nutrient intake may place strain on nutrient reserves. Nutrient scarcity, at the cellular and/or organismal level, promotes autophagy, a well-conserved subcellular catabolic process used to maintain energy homeostasis during periods of metabolic or hypoxic stress. An analysis of the putative alligator autophagy-related proteins has been conducted, and the results will be used to investigate the physiological role of autophagy during the brumation period. Using published genomic data, we have determined that autophagy is highly conserved, and alligator amino acid sequences exhibit a high percentage of identity with human homologs. Transcriptome analysis conducted using liver tissue derived from alligators confirmed the expression of one or more isoforms of each of the 34 autophagy initiation and elongation genes assayed. Five autophagy-related proteins (ATG5, ATG9A, BECN1, ATG16L1, and MAP1-LC3B), with functions spanning the major stages of autophagy, have been detected in alligator liver tissue by western blot analysis. In addition, ATG5 was detected in alligator liver tissue by immunohistochemistry. This is the first characterization of autophagy in crocodylians, and the first description of autophagy-related protein expression in whole blood.

Microbial Assemblage Dynamics Within the American Alligator Nesting Ecosystem: a Comparative Approach Across Ecological Scales.

Understanding the ecological processes that shape species assemblage patterns is central to community ecology. The effects of ecological processes on assemblage patterns are scale-dependent. We used metabarcoding and shotgun sequencing to determine bacterial taxonomic and functional assemblage patterns among varying defined focal scales (micro-, meso-, and macroscale) within the American alligator (Alligator mississippiensis) nesting microbiome. We correlate bacterial assemblage patterns among eight nesting compartments within and proximal to alligator nests (micro-), across 18 nests (meso-), and between 4 geographic sampling sites (macro-), to determine which ecological processes may drive bacterial assemblage patterns within the nesting environment. Among all focal scales, bacterial taxonomic and functional richness (α-diversity) did not statistically differ. In contrast, bacterial assemblage structure (ß-diversity) was unique across all focal scales, whereas functional pathways were redundant within nests and across geographic sites. Considering these observed scale-based patterns, taxonomic bacterial composition may be governed by unique environmental filters and dispersal limitations relative to microbial functional attributes within the alligator nesting environment. These results advance pattern-process dynamics within the field of microbial community ecology and describe processes influencing the American alligator nest microbiome.

Novel combinatorial screening identifies neurotrophic factors for selective classes of motor neurons.

Numerous neurotrophic factors promote the survival of developing motor neurons but their combinatorial actions remain poorly understood; to address this, we here screened 66 combinations of 12 neurotrophic factors on pure, highly viable, and standardized embryonic mouse motor neurons isolated by a unique FACS technique. We demonstrate potent, strictly additive, survival effects of hepatocyte growth factor (HGF), ciliary neurotrophic factor (CNTF), and Artemin through specific activation of their receptor complexes in distinct subsets of lumbar motor neurons: HGF supports hindlimb motor neurons through c-Met; CNTF supports subsets of axial motor neurons through CNTFRα; and Artemin acts as the first survival factor for parasympathetic preganglionic motor neurons through GFRα3/Syndecan-3 activation. These data show that neurotrophic factors can selectively promote the survival of distinct classes of embryonic motor neurons. Similar studies on postnatal motor neurons may provide a conceptual framework for the combined therapeutic use of neurotrophic factors in degenerative motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy, and spinobulbar muscular atrophy.

Mechanism of MEK inhibition determines efficacy in mutant KRAS- versus BRAF-driven cancers.

KRAS and BRAF activating mutations drive tumorigenesis through constitutive activation of the MAPK pathway. As these tumours represent an area of high unmet medical need, multiple allosteric MEK inhibitors, which inhibit MAPK signalling in both genotypes, are being tested in clinical trials. Impressive single-agent activity in BRAF-mutant melanoma has been observed; however, efficacy has been far less robust in KRAS-mutant disease. Here we show that, owing to distinct mechanisms regulating MEK activation in KRAS- versus BRAF-driven tumours, different mechanisms of inhibition are required for optimal antitumour activity in each genotype. Structural and functional analysis illustrates that MEK inhibitors with superior efficacy in KRAS-driven tumours (GDC-0623 and G-573, the former currently in phase I clinical trials) form a strong hydrogen-bond interaction with S212 in MEK that is critical for blocking MEK feedback phosphorylation by wild-type RAF. Conversely, potent inhibition of active, phosphorylated MEK is required for strong inhibition of the MAPK pathway in BRAF-mutant tumours, resulting in superior efficacy in this genotype with GDC-0973 (also known as cobimetinib), a MEK inhibitor currently in phase III clinical trials. Our study highlights that differences in the activation state of MEK in KRAS-mutant tumours versus BRAF-mutant tumours can be exploited through the design of inhibitors that uniquely target these distinct activation states of MEK. These inhibitors are currently being evaluated in clinical trials to determine whether improvements in therapeutic index within KRAS versus BRAF preclinical models translate to improved clinical responses in patients.

Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors.

Mutationally activated kinases define a clinically validated class of targets for cancer drug therapy. However, the efficacy of kinase inhibitors in patients whose tumours harbour such alleles is invariably limited by innate or acquired drug resistance. The identification of resistance mechanisms has revealed a recurrent theme­the engagement of survival signals redundant to those transduced by the targeted kinase. Cancer cells typically express multiple receptor tyrosine kinases (RTKs) that mediate signals that converge on common critical downstream cell-survival effectors­most notably, phosphatidylinositol-3-OH kinase (PI(3)K) and mitogen-activated protein kinase (MAPK). Consequently, an increase in RTK-ligand levels, through autocrine tumour-cell production, paracrine contribution from tumour stroma or systemic production, could confer resistance to inhibitors of an oncogenic kinase with a similar signalling output. Here, using a panel of kinase-'addicted' human cancer cell lines, we found that most cells can be rescued from drug sensitivity by simply exposing them to one or more RTK ligands. Among the findings with clinical implications was the observation that hepatocyte growth factor (HGF) confers resistance to the BRAF inhibitor PLX4032 (vemurafenib) in BRAF-mutant melanoma cells. These observations highlight the extensive redundancy of RTK-transduced signalling in cancer cells and the potentially broad role of widely expressed RTK ligands in innate and acquired resistance to drugs targeting oncogenic kinases.

Metabolite profiling stratifies pancreatic ductal adenocarcinomas into subtypes with distinct sensitivities to metabolic inhibitors.

Although targeting cancer metabolism is a promising therapeutic strategy, clinical success will depend on an accurate diagnostic identification of tumor subtypes with specific metabolic requirements. Through broad metabolite profiling, we successfully identified three highly distinct metabolic subtypes in pancreatic ductal adenocarcinoma (PDAC). One subtype was defined by reduced proliferative capacity, whereas the other two subtypes (glycolytic and lipogenic) showed distinct metabolite levels associated with glycolysis, lipogenesis, and redox pathways, confirmed at the transcriptional level. The glycolytic and lipogenic subtypes showed striking differences in glucose and glutamine utilization, as well as mitochondrial function, and corresponded to differences in cell sensitivity to inhibitors of glycolysis, glutamine metabolism, lipid synthesis, and redox balance. In PDAC clinical samples, the lipogenic subtype associated with the epithelial (classical) subtype, whereas the glycolytic subtype strongly associated with the mesenchymal (QM-PDA) subtype, suggesting functional relevance in disease progression. Pharmacogenomic screening of an additional ∼ 200 non-PDAC cell lines validated the association between mesenchymal status and metabolic drug response in other tumor indications. Our findings highlight the utility of broad metabolite profiling to predict sensitivity of tumors to a variety of metabolic inhibitors.

Tankyrase Inhibition Causes Reversible Intestinal Toxicity in Mice with a Therapeutic Index < 1.

Activated Wnt/ß-catenin signaling is frequently associated with colorectal cancer. Wnt inhibitors, including tankyrase inhibitors, are being explored as potential anticancer agents. Wnt signaling is also critical for intestinal tissue homeostasis, and Wnt inhibitors have been shown to cause intestinal toxicity in mice by affecting intestinal stem cells. This study sought to characterize the intestinal toxicity of tankyrase inhibitors, including reversibility, and to assess their therapeutic index. Novel tankyrase inhibitor G-631 caused dose-dependent intestinal toxicity with a therapeutic index < 1 after 14 days of dosing in mice. At a tolerated subtherapeutic dose level, the intestinal toxicity was composed of enteritis characterized by villus blunting, epithelial degeneration, and inflammation, which fully reversed after 14 days of recovery. Doubled exposure showed weak antitumor activity in a xenograft colorectal cancer model but also caused more severe intestinal toxicity characterized by multifocal-regionally extensive necrotizing and ulcerative enteritis leading to morbidity or moribundity in some animals. This toxicity was only partially reversed after 14 days of recovery, with evidence of crypt and villus regeneration, mildly blunted villi, and/or scarring in association with chronic inflammation of the submucosa. Therefore, the clinical utility of tankyrase inhibitors is likely limited by the on-target intestinal toxicity and a therapeutic index < 1 in mice.

Methyltestosterone alters sex determination in the American alligator (Alligator mississippiensis).

Effects of xenobiotics can be organizational, permanently affecting anatomy during embryonic development, and/or activational, influencing transitory actions during adulthood. The organizational influence of endocrine-disrupting contaminants (EDC's) produces a wide variety of reproductive abnormalities among vertebrates that exhibit temperature-dependent sex determination (TSD). Typically, such influences result in subsequent activational malfunction, some of which are beneficial in aquaculture. For example, 17-αmethyltestosterone (MT), a synthetic androgen, is utilized in tilapia farming to bias sex ratio towards males because they are more profitable. A heavily male-biased hatchling sex ratio is reported from a crocodile population near one such tilapia operation in Guanacaste, Costa Rica. In this study we test the effects of MT on sexual differentiation in American alligators, which we used as a surrogate for all crocodilians. Experimentally, alligators were exposed to MT in ovo at standard ecotoxicological concentrations. Sexual differentiation was determined by examination of primary and secondary sex organs post hatching. We find that MT is capable of producing male embryos at temperatures known to produce females and demonstrate a dose-dependent gradient of masculinization. Embryonic exposure to MT results in hermaphroditic primary sex organs, delayed renal development and masculinization of the clitero-penis (CTP).

Hepatocyte growth factor, a determinant of airspace homeostasis in the murine lung.

The alveolar compartment, the fundamental gas exchange unit in the lung, is critical for tissue oxygenation and viability. We explored hepatocyte growth factor (HGF), a pleiotrophic cytokine that promotes epithelial proliferation, morphogenesis, migration, and resistance to apoptosis, as a candidate mediator of alveolar formation and regeneration. Mice deficient in the expression of the HGF receptor Met in lung epithelial cells demonstrated impaired airspace formation marked by a reduction in alveolar epithelial cell abundance and survival, truncation of the pulmonary vascular bed, and enhanced oxidative stress. Administration of recombinant HGF to tight-skin mice, an established genetic emphysema model, attenuated airspace enlargement and reduced oxidative stress. Repair in the TSK/+ mouse was punctuated by enhanced akt and stat3 activation. HGF treatment of an alveolar epithelial cell line not only induced proliferation and scattering of the cells but also conferred protection against staurosporine-induced apoptosis, properties critical for alveolar septation. HGF promoted cell survival was attenuated by akt inhibition. Primary alveolar epithelial cells treated with HGF showed improved survival and enhanced antioxidant production. In conclusion, using both loss-of-function and gain-of-function maneuvers, we show that HGF signaling is necessary for alveolar homeostasis in the developing lung and that augmentation of HGF signaling can improve airspace morphology in murine emphysema. Our studies converge on prosurvival signaling and antioxidant protection as critical pathways in HGF-mediated airspace maintenance or repair. These findings support the exploration of HGF signaling enhancement for diseases of the airspace.

Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent.

Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF ß-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not ß-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.

PAK1 mediates pancreatic cancer cell migration and resistance to MET inhibition.

Pancreatic adenocarcinoma (PDAC) is a major unmet medical need and a deeper understanding of molecular drivers is needed to advance therapeutic options for patients. We report here that p21-activated kinase 1 (PAK1) is a central node in PDAC cells downstream of multiple growth factor signalling pathways, including hepatocyte growth factor (HGF) and MET receptor tyrosine kinase. PAK1 inhibition blocks signalling to cytoskeletal effectors and tumour cell motility driven by HGF/MET. MET antagonists, such as onartuzumab and crizotinib, are currently in clinical development. Given that even highly effective therapies have resistance mechanisms, we show that combination with PAK1 inhibition overcomes potential resistance mechanisms mediated either by activation of parallel growth factor pathways or by direct amplification of PAK1. Inhibition of PAK1 attenuated in vivo tumour growth and metastasis in a model of pancreatic adenocarcinoma. In human tissues, PAK1 is highly expressed in a proportion of PDACs (33% IHC score 2 or 3; n = 304) and its expression is significantly associated with MET positivity (p < 0.0001) and linked to a widespread metastatic pattern in patients (p = 0.067). Taken together, our results provide evidence for a functional role of MET/PAK1 signalling in pancreatic adenocarcinoma and support further characterization of therapeutic inhibitors in this indication.

Network quantification of EGFR signaling unveils potential for targeted combination therapy.

The epidermal growth factor receptor (EGFR) signaling network is activated in most solid tumors, and small-molecule drugs targeting this network are increasingly available. However, often only specific combinations of inhibitors are effective. Therefore, the prediction of potent combinatorial treatments is a major challenge in targeted cancer therapy. In this study, we demonstrate how a model-based evaluation of signaling data can assist in finding the most suitable treatment combination. We generated a perturbation data set by monitoring the response of RAS/PI3K signaling to combined stimulations and inhibitions in a panel of colorectal cancer cell lines, which we analyzed using mathematical models. We detected that a negative feedback involving EGFR mediates strong cross talk from ERK to AKT. Consequently, when inhibiting MAPK, AKT activity is increased in an EGFR-dependent manner. Using the model, we predict that in contrast to single inhibition, combined inactivation of MEK and EGFR could inactivate both endpoints of RAS, ERK and AKT. We further could demonstrate that this combination blocked cell growth in BRAF- as well as KRAS-mutated tumor cells, which we confirmed using a xenograft model.

Constitutive Innate Immunity and Systemic Responses to Infection of the American Alligator (Alligator mississippiensis).

Uninfected alligators (Alligator mississippiensis) exhibited high constitutive levels of hepatic gene expression related to immune function, whereas the highest-expressed hepatic genes of uninfected mice were related to metabolism. Intraperitoneal challenge of mice with bacterial lipopolysaccharide results in dramatic inflammatory effects including peritoneal ascites, febrile response, dramatic alterations in electrophoretic serum profile, and mortality. In contrast, coelomic injection of alligators with 200× the murine LD50 of intraperitoneal bacterial lipopolysaccharide resulted in no changes in serum protein profiles, behavioral effects, mortality, and no coelomic ascites. However, injection of juvenile alligators with live bacteria resulted in a titer-dependent decrease in metabolic rate, as measured by oxygen consumption. These results are the opposite of those observed for mammalian and avian species. The decreased oxygen consumption was not accompanied by changes in heart or respiration rate, indicating that this phenomenon was not due to bradycardia or bradypnea. Interestingly, challenge of alligators with bacteria resulted in the complete expulsion of digestive tract contents within four hours. We interpret these activities as temporary minimization of other biological systemic activities to redirect and devote energy to immune function. The reallocation of resources within an organism to fight infection without increases in metabolic rate has not been described in other animals.

PROTACs Targeting BRM (SMARCA2) Afford Selective In Vivo Degradation over BRG1 (SMARCA4) and Are Active in BRG1 Mutant Xenograft Tumor Models.

The identification of VHL-binding proteolysis targeting chimeras (PROTACs) that potently degrade the BRM protein (also known as SMARCA2) in SW1573 cell-based experiments is described. These molecules exhibit between 10- and 100-fold degradation selectivity for BRM over the closely related paralog protein BRG1 (SMARCA4). They also selectively impair the proliferation of the H1944 "BRG1-mutant" NSCLC cell line, which lacks functional BRG1 protein and is thus highly dependent on BRM for growth, relative to the wild-type Calu6 line. In vivo experiments performed with a subset of compounds identified PROTACs that potently and selectively degraded BRM in the Calu6 and/or the HCC2302 BRG1 mutant NSCLC xenograft models and also afforded antitumor efficacy in the latter system. Subsequent PK/PD analysis established a need to achieve strong BRM degradation (>95%) in order to trigger meaningful antitumor activity in vivo. Intratumor quantitation of mRNA associated with two genes whose transcription was controlled by BRM (PLAU and KRT80) also supported this conclusion.

Pharmacokinetic drivers of toxicity for basic molecules: strategy to lower pKa results in decreased tissue exposure and toxicity for a small molecule Met inhibitor.

Several toxicities are clearly driven by free drug concentrations in plasma, such as toxicities related to on-target exaggerated pharmacology or off-target pharmacological activity associated with receptors, enzymes or ion channels. However, there are examples in which organ toxicities appear to correlate better with total drug concentrations in the target tissues, rather than with free drug concentrations in plasma. Here we present a case study in which a small molecule Met inhibitor, GEN-203, with significant liver and bone marrow toxicity in preclinical species was modified with the intention of increasing the safety margin. GEN-203 is a lipophilic weak base as demonstrated by its physicochemical and structural properties: high LogD (distribution coefficient) (4.3) and high measured pKa (7.45) due to the basic amine (N-ethyl-3-fluoro-4-aminopiperidine). The physicochemical properties of GEN-203 were hypothesized to drive the high distribution of this compound to tissues as evidenced by a moderately-high volume of distribution (Vd>3l/kg) in mouse and subsequent toxicities of the compound. Specifically, the basicity of GEN-203 was decreased through addition of a second fluorine in the 3-position of the aminopiperidine to yield GEN-890 (N-ethyl-3,3-difluoro-4-aminopiperidine), which decreased the volume of distribution of the compound in mouse (Vd=1.0l/kg), decreased its tissue drug concentrations and led to decreased toxicity in mice. This strategy suggests that when toxicity is driven by tissue drug concentrations, optimization of the physicochemical parameters that drive tissue distribution can result in decreased drug concentrations in tissues, resulting in lower toxicity and improved safety margins.