Targeting fatty acid synthase in preclinical models of TNBC brain metastases synergizes with SN-38 and impairs invasion.

Fatty acid synthesis (FAS) has been shown to play a key role in the survival of brain-metastatic (BM) breast cancer. We demonstrate that the fatty acid synthase inhibitor TVB-2640 synergizes with the topoisomerase inhibitor SN-38 in triple-negative breast cancer (TNBC) BM cell lines, upregulates FAS and downregulates cell cycle progression gene expression, and slows the motility of TNBC BM cell lines. The combination of SN-38 and TVB-2640 warrants further consideration as a potential therapeutic option in TNBC BMs.

Selective and Potent PROTAC Degraders of c-Src Kinase.

Using dasatinib linked to E3 ligase ligands, we identified a potent and selective dual Csk/c-Src PROTAC degrader. We then replaced dasatinib, the c-Src-directed ligand, with a conformation-selective analogue that stabilizes the αC-helix-out conformation of c-Src. Using the αC-helix-out ligand, we identified a PROTAC that is potent and selective for c-Src. We demonstrated a high degree of catalysis with our c-Src PROTACs. Using our c-Src PROTACs, we identified pharmacological advantages of c-Src degradation compared to inhibition with respect to cancer cell proliferation.

Optimizing Precision Medicine for Breast Cancer Brain Metastases with Functional Drug Response Assessment.

The development of novel therapies for brain metastases is an unmet need. Brain metastases may have unique molecular features that could be explored as therapeutic targets. A better understanding of the drug sensitivity of live cells coupled to molecular analyses will lead to a rational prioritization of therapeutic candidates. We evaluated the molecular profiles of 12 breast cancer brain metastases (BCBM) and matched primary breast tumors to identify potential therapeutic targets. We established six novel patient-derived xenograft (PDX) from BCBM from patients undergoing clinically indicated surgical resection of BCBM and used the PDXs as a drug screening platform to interrogate potential molecular targets. Many of the alterations were conserved in brain metastases compared with the matched primary. We observed differential expressions in the immune-related and metabolism pathways. The PDXs from BCBM captured the potentially targetable molecular alterations in the source brain metastases tumor. The alterations in the PI3K pathway were the most predictive for drug efficacy in the PDXs. The PDXs were also treated with a panel of over 350 drugs and demonstrated high sensitivity to histone deacetylase and proteasome inhibitors. Our study revealed significant differences between the paired BCBM and primary breast tumors with the pathways involved in metabolisms and immune functions. While molecular targeted drug therapy based on genomic profiling of tumors is currently evaluated in clinical trials for patients with brain metastases, a functional precision medicine strategy may complement such an approach by expanding potential therapeutic options, even for BCBM without known targetable molecular alterations. Significance: Examining genomic alterations and differentially expressed pathways in brain metastases may inform future therapeutic strategies. This study supports genomically-guided therapy for BCBM and further investigation into incorporating real-time functional evaluation will increase confidence in efficacy estimations during drug development and predictive biomarker assessment for BCBM.

TMPRSS2 Inhibitor Discovery Facilitated through an In Silico and Biochemical Screening Platform.

The COVID-19 pandemic has highlighted the need for new antiviral approaches because many of the currently approved drugs have proven ineffective against mitigating SARS-CoV-2 infections. The host transmembrane serine protease TMPRSS2 is a promising antiviral target because it plays a role in priming the spike protein before viral entry occurs for the most virulent variants. Further, TMPRSS2 has no established physiological role, thereby increasing its attractiveness as a target for antiviral agents. Here, we utilize virtual screening to curate large libraries into a focused collection of potential inhibitors. Optimization of a recombinant expression and purification protocol for the TMPRSS2 peptidase domain facilitates subsequent biochemical screening and characterization of selected compounds from the curated collection in a kinetic assay. In doing so, we identify new noncovalent TMPRSS2 inhibitors that block SARS-CoV-2 infectivity in a cellular model. One such inhibitor, debrisoquine, has high ligand efficiency, and an initial structure-activity relationship study demonstrates that debrisoquine is a tractable hit compound for TMPRSS2.

QSAR via multisite λ-dynamics in the orphaned TSSK1B kinase.

Multisite λ-dynamics (MSλD) is a novel method for the calculation of relative free energies of binding for ligands to their targeted receptors. It can be readily used to examine a large number of molecules with multiple functional groups at multiple sites around a common core. This makes MSλD a powerful tool in structure-based drug design. In the present study, MSλD is applied to calculate the relative binding free energies of 1296 inhibitors to the testis specific serine kinase 1B (TSSK1B), a validated target for male contraception. For this system, MSλD requires significantly fewer computational resources compared to traditional free energy methods like free energy perturbation or thermodynamic integration. From MSλD simulations, we examined whether modifications of a ligand at two different sites are coupled or not. Based on our calculations, we established a quantitative structure-activity relationship (QSAR) for this set of molecules and identified a site in the ligand where further modification, such as adding more polar groups, may lead to increased binding affinity.

Reply to Correspondence on "Synergy and Antagonism between Allosteric and Active-Site Inhibitors of Abl Tyrosine Kinase".

Manley and co-workers provide data demonstrating that, at super-pharmacological concentrations (300 µM), a ternary complex between Abl, asciminib, and ATP-competitive inhibitors is possible. The work in our manuscript concerns the interplay of asciminib (and GNF-2) with ATP-competitive inhibitors at pharmacologically relevant concentrations (Cmax =1.6-3.7 µM for asciminib). Manley and co-workers do not question any of the studies that we reported, nor do they provide explanations for how our work fits into their preferred model. Herein, we consider the data presented by Manley and co-workers. In addition, we provide new data supporting the findings in our Communication. Asciminib and ATP-competitive inhibitors do not simultaneously bind Abl at pharmacologically relevant concentrations unless the conformation selectivity for both ligands is matched.

Insights into the modular design of kinase inhibitors and application to Abl and Axl.

Scaffold hopping is a common strategy for generating kinase inhibitors that bind to the DFG-out inactive conformation. Small structural differences in inhibitor scaffolds can have significant effects on potency and selectivity across the kinome, however, these effects are often not studied in detail. Herein, we outline a design strategy to generate an array of DFG-out conformation inhibitors with three different hinge-binders and two DFG-pocket groups. We studied inhibitor selectivity across a large segment of the kinome and elucidated binding preferences that can be used in scaffold hopping campaigns. Using these analyses, we identified two selective inhibitors that display low nanomolar potency against Axl or wild-type and clinically relevant mutants of Abl.

Conformation-tunable ATP-competitive kinase inhibitors.

Small molecule kinase inhibitors have shown immense clinical utility for diverse indications. While >60 kinase inhibitors have been approved (and many more in clinical trials), it remains unclear whether the clinical efficacy of a kinase inhibitor is solely dependent on enzymatic inhibition, or whether non-catalytic functions play a role in the efficacy of some kinase inhibitors. Here, we designed and synthesized a series of pyrazolopyrimidine kinase inhibitors that modulate the global kinase conformation of c-Src kinase. Expanding upon our findings from the pyrazolopyrimidine inhibitor series, we designed, synthesized, and evaluated three pair of conformation-selective kinase inhibitors, each with a unique hinge-binding scaffold. We profiled each pair of kinase inhibitors across 468 kinases and identified 38 kinases that could be studied using these pair of conformation-selective inhibitors. We also explore the binding of conformation-selective kinase inhibitors to mutant kinases of EGFR, FLT3, and KIT. Together, these studies yield important insight into the design of conformation-tunable kinase inhibitors and provide a toolset of compounds to study the role of protein conformation on kinase signaling.

Synergy and Antagonism between Allosteric and Active-Site Inhibitors of Abl Tyrosine Kinase.

Allosteric inhibitors of Abl kinase are being explored in the clinic, often in combination with ATP-site inhibitors of Abl kinase. However, there are conflicting data on whether both ATP-competitive inhibitors and myristoyl-site allosteric inhibitors can simultaneously bind Abl kinase. Here, we determine whether there is synergy or antagonism between ATP-competitive inhibitors and allosteric inhibitors of Abl. We observe that clinical ATP-competitive inhibitors are not synergistic with allosteric ABL inhibitors, however, conformation-selective ATP-site inhibitors that modulate the global conformation of Abl can afford synergy. We demonstrate that kinase conformation is the key driver to simultaneously bind two compounds to Abl kinase. Finally, we explore the interaction of allosteric and conformation selective ATP-competitive inhibitors in a series of biochemical and cellular assays.

TMPRSS2 inhibitor discovery facilitated through an in silico and biochemical screening platform.

The COVID-19 pandemic has highlighted the need for new antiviral targets, as many of the currently approved drugs have proven ineffective against mitigating SARS-CoV-2 infections. The host transmembrane serine protease TMPRSS2 is a highly promising antiviral target, as it plays a direct role in priming the spike protein before viral entry occurs. Further, unlike other targets such as ACE2, TMPRSS2 has no known biological role. Here we utilize virtual screening to curate large libraries into a focused collection of potential inhibitors. Optimization of a recombinant expression and purification protocol for the TMPRSS2 peptidase domain facilitates subsequent biochemical screening and characterization of selected compounds from the curated collection in a kinetic assay. In doing so, we demonstrate that serine protease inhibitors camostat, nafamostat, and gabexate inhibit through a covalent mechanism. We further identify new non-covalent compounds as TMPRSS2 protease inhibitors, demonstrating the utility of a combined virtual and experimental screening campaign in rapid drug discovery efforts.

Scribble sub-cellular localization modulates recruitment of YES1 to regulate YAP1 phosphorylation.

The multi-domain scaffolding protein Scribble (Scrib) regulates cell polarity and growth signaling at cell-cell junctions. In epithelial cancers, Scrib mislocalization and overexpression paradoxically transform Scrib from a basolateral tumor suppressor to a cytosolic driver of tumorigenicity. To address the function of Scrib (mis)localization, a Scrib-HaloTag fusion was genome engineered in polarized epithelial cells. Expression of the epithelial to mesenchymal transcription factor Snail displaced Scrib-HaloTag from cell junctions, mirroring the mislocalization observed in cancers. Interestingly, Snail expression promotes Yes-associated protein-1 (YAP1) nuclear localization independent of hippo pathway-regulated YAP-S127 phosphorylation. Furthermore, Scrib HaloPROTAC degradation attenuates YAP1-Y357 phosphorylation. Halo-ligand affinity purification mass spectrometry analysis identified the Src family kinase YES1 as a mislocalized Scrib interaction partner, preferentially recruiting the kinase active and open global conformation (αC helix in). Altogether, mislocalized Scrib enhances YAP1 phosphorylation by scaffolding active YES1.

Impact of between-tissue differences on pan-cancer predictions of drug sensitivity.

Increased availability of drug response and genomics data for many tumor cell lines has accelerated the development of pan-cancer prediction models of drug response. However, it is unclear how much between-tissue differences in drug response and molecular characteristics may contribute to pan-cancer predictions. Also unknown is whether the performance of pan-cancer models could vary by cancer type. Here, we built a series of pan-cancer models using two datasets containing 346 and 504 cell lines, each with MEK inhibitor (MEKi) response and mRNA expression, point mutation, and copy number variation data, and found that, while the tissue-level drug responses are accurately predicted (between-tissue ρ = 0.88-0.98), only 5 of 10 cancer types showed successful within-tissue prediction performance (within-tissue ρ = 0.11-0.64). Between-tissue differences make substantial contributions to the performance of pan-cancer MEKi response predictions, as exclusion of between-tissue signals leads to a decrease in Spearman's ρ from a range of 0.43-0.62 to 0.30-0.51. In practice, joint analysis of multiple cancer types usually has a larger sample size, hence greater power, than for one cancer type; and we observe that higher accuracy of pan-cancer prediction of MEKi response is almost entirely due to the sample size advantage. Success of pan-cancer prediction reveals how drug response in different cancers may invoke shared regulatory mechanisms despite tissue-specific routes of oncogenesis, yet predictions in different cancer types require flexible incorporation of between-cancer and within-cancer signals. As most datasets in genome sciences contain multiple levels of heterogeneity, careful parsing of group characteristics and within-group, individual variation is essential when making robust inference.

MEK is a promising target in the basal subtype of bladder cancer.

While many resources exist for the drug screening of bladder cancer cell lines in 2D culture, it is widely recognized that screening in 3D culture is more representative of in vivo response. Importantly, signaling changes between 2D and 3D culture can result in changes to drug response. To address the need for 3D drug screening of bladder cancer cell lines, we screened 17 bladder cancer cell lines using a library of 652 investigational small-molecules and 3 clinically relevant drug combinations in 3D cell culture. Our goal was to identify compounds and classes of compounds with efficacy in bladder cancer. Utilizing established genomic and transcriptomic data for these bladder cancer cell lines, we correlated the genomic molecular parameters with drug response, to identify potentially novel groups of tumors that are vulnerable to specific drugs or classes of drugs. Importantly, we demonstrate that MEK inhibitors are a promising targeted therapy for the basal subtype of bladder cancer, and our data indicate that drug screening of 3D cultures provides an important resource for hypothesis generation.

Molecular determinants of drug response in TNBC cell lines.

PURPOSE: There is a need for biomarkers of drug efficacy for targeted therapies in triple-negative breast cancer (TNBC). As a step toward this, we identify multi-omic molecular determinants of anti-TNBC efficacy in cell lines for a panel of oncology drugs. METHODS: Using 23 TNBC cell lines, drug sensitivity scores (DSS3) were determined using a panel of investigational drugs and drugs approved for other indications. Molecular readouts were generated for each cell line using RNA sequencing, RNA targeted panels, DNA sequencing, and functional proteomics. DSS3 values were correlated with molecular readouts using a FDR-corrected significance cutoff of p* < 0.05 and yielded molecular determinant panels that predict anti-TNBC efficacy. RESULTS: Six molecular determinant panels were obtained from 12 drugs we prioritized based on their efficacy. Determinant panels were largely devoid of DNA mutations of the targeted pathway. Molecular determinants were obtained by correlating DSS3 with molecular readouts. We found that co-inhibiting molecular correlate pathways leads to robust synergy across many cell lines. CONCLUSIONS: These findings demonstrate an integrated method to identify biomarkers of drug efficacy in TNBC where DNA predictions correlate poorly with drug response. Our work outlines a framework for the identification of novel molecular determinants and optimal companion drugs for combination therapy based on these correlates.

Selective Proteolysis to Study the Global Conformation and Regulatory Mechanisms of c-Src Kinase.

Protein kinase pathways are traditionally mapped by monitoring downstream phosphorylation. Meanwhile, the noncatalytic functions of protein kinases remain under-appreciated as critical components of kinase signaling. c-Src is a protein kinase known to have noncatalytic signaling function important in healthy and disease cell signaling. Large conformational changes in the regulatory domains regulate c-Src's noncatalytic functions. Herein, we demonstrate that changes in the global conformation of c-Src can be monitored using a selective proteolysis methodology. Further, we use this methodology to investigate changes in the global conformation of several clinical and nonclinical mutations of c-Src. Significantly, we identify a novel activating mutation observed clinically, W121R, that can escape down-regulation mechanisms. Our methodology can be expanded to monitor the global conformation of other tyrosine kinases, including c-Abl, and represents an important tool toward the elucidation of the noncatalytic functions of protein kinases.

IL-4/IL-13 Stimulated Macrophages Enhance Breast Cancer Invasion Via Rho-GTPase Regulation of Synergistic VEGF/CCL-18 Signaling.

Tumor associated macrophages (TAMs) are increasingly recognized as major contributors to the metastatic progression of breast cancer and enriched levels of TAMs often correlate with poor prognosis. Despite our current advances it remains unclear which subset of M2-like macrophages have the highest capacity to enhance the metastatic program and which mechanisms regulate this process. Effective targeting of macrophages that aid cancer progression requires knowledge of the specific mechanisms underlying their pro-metastatic actions, as to avoid the anticipated toxicities from generalized targeting of macrophages. To this end, we set out to understand the relationship between the regulation of tumor secretions by Rho-GTPases, which were previously demonstrated to affect them, macrophage differentiation, and the converse influence of macrophages on cancer cell phenotype. Our data show that IL-4/IL-13 in vitro differentiated M2a macrophages significantly increase migratory and invasive potential of breast cancer cells at a greater rate than M2b or M2c macrophages. Our previous work demonstrated that the Rho-GTPases are potent regulators of macrophage-induced migratory responses; therefore, we examined M2a-mediated responses in RhoA or RhoC knockout breast cancer cell models. We find that both RhoA and RhoC regulate migration and invasion in MDA-MB-231 and SUM-149 cells following stimulation with M2a conditioned media. Secretome analysis of M2a conditioned media reveals high levels of vascular endothelial growth factor (VEGF) and chemokine (C-C motif) ligand 18 (CCL-18). Results from our functional assays reveal that M2a TAMs synergistically utilize VEGF and CCL-18 to promote migratory and invasive responses. Lastly, we show that pretreatment with ROCK inhibitors Y-276332 or GSK42986A attenuated VEGF/CCL-18 and M2a-induced migration and invasion. These results support Rho-GTPase signaling regulates downstream responses induced by TAMs, offering a novel approach for the prevention of breast cancer metastasis by anti-RhoA/C therapies.

Bivalent Inhibitors of c-Src Tyrosine Kinase That Bind a Regulatory Domain.

We have developed a general methodology to produce bivalent kinase inhibitors for c-Src that interact with the SH2 and ATP binding pockets. Our approach led to a highly selective bivalent inhibitor of c-Src. We demonstrate impressive selectivity for c-Src over homologous kinases. Exploration of the unexpected high level of selectivity yielded insight into the inherent flexibility of homologous kinases. Finally, we demonstrate that our methodology is modular and both the ATP-competitive fragment and conjugation chemistry can be swapped.

UM-164: A Potent c-Src/p38 Kinase Inhibitor with In Vivo Activity against Triple-Negative Breast Cancer.

PURPOSE: c-Src has been shown to play a pivotal role in breast cancer progression, metastasis, and angiogenesis. In the clinic, however, the limited efficacy and high toxicity of existing c-Src inhibitors have tempered the enthusiasm for targeting c-Src. We developed a novel c-Src inhibitor (UM-164) that specifically binds the DFG-out inactive conformation of its target kinases. We hypothesized that binding the inactive kinase conformation would lead to improved pharmacologic outcomes by altering the noncatalytic functions of the targeted kinases. EXPERIMENTAL DESIGN: We have analyzed the anti-triple-negative breast cancer (TNBC) activity of UM-164 in a comprehensive manner that includes in vitro cell proliferation, migration, and invasion assays (including a novel patient-derived xenograft cell line, VARI-068), along with in vivo TNBC xenografts. RESULTS: We demonstrate that UM-164 binds the inactive kinase conformation of c-Src. Kinome-wide profiling of UM-164 identified that Src and p38 kinase families were potently inhibited by UM-164. We further demonstrate that dual c-Src/p38 inhibition is superior to mono-inhibition of c-Src or p38 alone. We demonstrate that UM-164 alters the cell localization of c-Src in TNBC cells. In xenograft models of TNBC, UM-164 resulted in a significant decrease of tumor growth compared with controls, with limited in vivo toxicity. CONCLUSIONS: In contrast with c-Src kinase inhibitors used in the clinic (1, 2), we demonstrate in vivo efficacy in xenograft models of TNBC. Our results suggest that the dual activity drug UM-164 is a promising lead compound for developing the first targeted therapeutic strategy against TNBC. Clin Cancer Res; 22(20); 5087-96. ©2016 AACR.

Conformation-Selective Analogues of Dasatinib Reveal Insight into Kinase Inhibitor Binding and Selectivity.

In the kinase field, there are many widely held tenets about conformation-selective inhibitors that have yet to be validated using controlled experiments. We have designed, synthesized, and characterized a series of kinase inhibitor analogues of dasatinib, an FDA-approved kinase inhibitor that binds the active conformation. This inhibitor series includes two Type II inhibitors that bind the DFG-out inactive conformation and two inhibitors that bind the αC-helix-out inactive conformation. Using this series of compounds, we analyze the impact that conformation-selective inhibitors have on target binding and kinome-wide selectivity.