Activating point mutations in the MET kinase domain represent a unique molecular subset of lung cancer and other malignancies targetable with MET inhibitors.

Activating point mutations in the MET tyrosine kinase domain (TKD) are oncogenic in a subset of papillary renal cell carcinomas (PRCC). Here, using comprehensive genomic profiling among >600,000 patients, we identify activating MET TKD point mutations as putative oncogenic driver across diverse cancers, with a frequency of ~0.5%. The most common mutations in the MET TKD defined as oncogenic or likely oncogenic according to OncoKB resulted in amino acid substitutions at positions H1094, L1195, F1200, D1228, Y1230, M1250, and others. Preclinical modeling of these alterations confirmed their oncogenic potential, and also demonstrated differential patterns of sensitivity to type I and type II MET inhibitors. Two patients with metastatic lung adenocarcinoma harboring MET TKD mutations (H1094Y, F1200I) and no other known oncogenic drivers achieved confirmed partial responses to a type I MET inhibitor. Activating MET TKD mutations occur in multiple malignancies and may confer clinical sensitivity to currently available MET inhibitors.

Parsing digital or analogue TCR performance through piconewton forces.

αß T-cell receptors (TCRs) recognize aberrant peptides bound to major histocompatibility complex molecules (pMHCs) on unhealthy cells, amplifying specificity and sensitivity through physical load placed on the TCR-pMHC bond during immunosurveillance. To understand this mechanobiology, TCRs stimulated by abundantly and sparsely arrayed epitopes (NP 366-374 /D b and PA 224-233 /D b , respectively) following in vivo influenza A virus infection were studied with optical tweezers. While certain NP repertoire CD8 T lymphocytes require many ligands for activation, others are digital, needing just few. Conversely, all PA TCRs perform digitally, exhibiting pronounced bond lifetime increases through sustained, energizing volleys of structural transitioning. Optimal digital performance is superior in vivo, correlating with ERK phosphorylation, CD3 loss, and activation marker upregulation in vitro . Given neoantigen array paucity, digital TCRs are likely critical for immunotherapies. One Sentence Summary: Quality of ligand recognition in a T-cell repertoire is revealed through application of physical load on clonal T-cell receptor (TCR)-pMHC bonds.

Hepatic Huwe1 loss protects mice from non-alcoholic fatty liver disease through lipid metabolic rewiring.

Non-alcoholic fatty liver disease (NAFLD) is the most pervasive liver pathology worldwide. Here, we demonstrate that the ubiquitin E3 ligase Huwe1 is vital in NAFLD pathogenesis. Using mass spectrometry and RNA sequencing, we reveal that liver-specific deletion of Huwe1 (Huwe1LKO) in 1-year-old mice (approximately middle age in humans) elicits extensive lipid metabolic reprogramming that involves downregulation of de novo lipogenesis and fatty acid uptake, upregulation of fatty acid ß-oxidation, and increased oxidative phosphorylation. ChEA transcription factor prediction analysis inferred these changes result from attenuated PPARɑ, LXR, and RXR activity in Huwe1LKO livers. Consequently, Huwe1LKO mice fed chow diet exhibited significantly reduced hepatic steatosis and superior glucose tolerance compared to wild-type mice. Huwe1LKO also conferred protection from high-fat diet-induced hepatic steatosis by 6-months of age, with increasingly robust differences observed as mice reached middle age. Together, we present evidence that Huwe1 plays a critical role in the development of age- and diet-induced NAFLD.

Mammalian SWI/SNF chromatin remodeling complexes promote tyrosine kinase inhibitor resistance in EGFR-mutant lung cancer.

Acquired resistance to tyrosine kinase inhibitors (TKI), such as osimertinib used to treat EGFR-mutant lung adenocarcinomas, limits long-term efficacy and is frequently caused by non-genetic mechanisms. Here, we define the chromatin accessibility and gene regulatory signatures of osimertinib sensitive and resistant EGFR-mutant cell and patient-derived models and uncover a role for mammalian SWI/SNF chromatin remodeling complexes in TKI resistance. By profiling mSWI/SNF genome-wide localization, we identify both shared and cancer cell line-specific gene targets underlying the resistant state. Importantly, genetic and pharmacologic disruption of the SMARCA4/SMARCA2 mSWI/SNF ATPases re-sensitizes a subset of resistant models to osimertinib via inhibition of mSWI/SNF-mediated regulation of cellular programs governing cell proliferation, epithelial-to-mesenchymal transition, epithelial cell differentiation, and NRF2 signaling. These data highlight the role of mSWI/SNF complexes in supporting TKI resistance and suggest potential utility of mSWI/SNF inhibitors in TKI-resistant lung cancers.

The Role of CD36 in Cancer Progression and Its Value as a Therapeutic Target.

Cluster of differentiation 36 (CD36) is a cell surface scavenger receptor that plays critical roles in many different types of cancer, notably breast, brain, and ovarian cancers. While it is arguably most well-known for its fatty acid uptake functions, it is also involved in regulating cellular adhesion, immune response, and apoptosis depending on the cellular and environmental contexts. Here, we discuss the multifaceted role of CD36 in cancer biology, such as its role in mediating metastasis, drug resistance, and immune evasion to showcase its potential as a therapeutic target. We will also review existing approaches to targeting CD36 in pre-clinical studies, as well as discuss the only CD36-targeting drug to advance to late-stage clinical trials, VT1021. Given the roles of CD36 in the etiology of metabolic disorders, such as atherosclerosis, diabetes, and non-alcoholic fatty liver disease, the clinical implications of CD36-targeted therapy are wide-reaching, even beyond cancer.

Macrocyclization of Quinazoline-Based EGFR Inhibitors Leads to Exclusive Mutant Selectivity for EGFR L858R and Del19.

Activating mutations in the epidermal growth factor receptor (EGFR) are frequent oncogenic drivers of non-small-cell lung cancer (NSCLC). The most frequent alterations in EGFR are short in-frame deletions in exon 19 (Del19) and the missense mutation L858R, which both lead to increased activity and sensitization of NSCLC to EGFR inhibition. The first approved EGFR inhibitors used for first-line treatment of NSCLC, gefitinib and erlotinib, are quinazoline-based. However, both inhibitors have several known off-targets, and they also potently inhibit wild-type (WT) EGFR, resulting in side effects. Here, we applied a macrocyclic strategy on a quinazoline-based scaffold as a proof-of-concept study with the goal of increasing kinome-wide selectivity of this privileged inhibitor scaffold. Kinome-wide screens and SAR studies yielded 3f, a potent inhibitor for the most common EGFR mutation (EGFR Del19: 119 nM) with selectivity against the WT receptor (EGFR: >10 µM) and the kinome.

An allosteric inhibitor against the therapy-resistant mutant forms of EGFR in non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) therapy using small-molecule tyrosine kinase inhibitors (TKIs) is initially efficacious in patients with EGFR-mutant lung cancer, although drug resistance eventually develops. Allosteric EGFR inhibitors, which bind to a different EGFR site than existing ATP-competitive EGFR TKIs, have been developed as a strategy to overcome therapy-resistant EGFR mutations. Here we identify and characterize JBJ-09-063, a mutant-selective allosteric EGFR inhibitor that is effective across EGFR TKI-sensitive and resistant models, including those with EGFR T790M and C797S mutations. We further uncover that EGFR homo- or heterodimerization with other ERBB family members, as well as the EGFR L747S mutation, confers resistance to JBJ-09-063, but not to ATP-competitive EGFR TKIs. Overall, our studies highlight the potential clinical utility of JBJ-09-063 as a single agent or in combination with EGFR TKIs to define more effective strategies to treat EGFR-mutant lung cancer.

X-Linked Huwe1 Is Essential for Oocyte Maturation and Preimplantation Embryo Development.

HUWE1 is a HECT-domain ubiquitin E3 ligase expressed in various tissues. Although HUWE1 is known to promote degradation of the tumor suppressor p53, given a growing list of its substrates, in vivo functions of HUWE1 remain elusive. Here, we investigated the role of HUWE1 in the female reproductive system. Homozygous deletion of Huwe1 in mouse oocytes of primary follicles caused oocyte death and female infertility, whereas acute depletion of HUWE1 protein by Trim-Away technology did not impact oocytes from antral follicles. Interestingly, oocytes from Huwe1 heterozygous females matured and fertilized normally, but the majority of embryos that lacked maternal Huwe1 were arrested at the morula stage after fertilization. Consequently, Huwe1 heterozygous females only produced wild-type pups. Concomitant knockout of p53 did not recover fertility of the Huwe1 knockout females. These findings make HUWE1 a unique and critical maternal factor indispensable for maintaining the quality of oocytes and embryos.

Lipid metabolic reprogramming as an emerging mechanism of resistance to kinase inhibitors in breast cancer.

Breast cancer is one of the leading causes of death in women in the United States. In general, patients with breast cancer undergo surgical resection of the tumor and/or receive drug treatment to kill or suppress the growth of cancer cells. In this regard, small molecule kinase inhibitors serve as an important class of drugs used in clinical and research settings. However, the development of resistance to these compounds, in particular HER2 and CDK4/6 inhibitors, often limits durable clinical responses to therapy. Emerging evidence indicates that PI3K/AKT/mTOR pathway hyperactivation is one of the most prominent mechanisms of resistance to many small molecule inhibitors as it bypasses upstream growth factor receptor inhibition. Importantly, the PI3K/AKT/mTOR pathway also plays a pertinent role in regulating various aspects of cancer metabolism. Recent studies from our lab and others have demonstrated that altered lipid metabolism mediates the development of acquired drug resistance to HER2-targeted therapies in breast cancer, raising an interesting link between reprogrammed kinase signaling and lipid metabolism. It appears that, upon development of resistance to HER2 inhibitors, breast cancer cells rewire lipid metabolism to somehow circumvent the inhibition of kinase signaling. Here, we review various mechanisms of resistance observed for kinase inhibitors and discuss lipid metabolism as a potential therapeutic target to overcome acquired drug resistance.

CD36: a key mediator of resistance to HER2 inhibitors in breast cancer.

Acquired resistance to anti-HER2 therapy is a significant clinical challenge in breast cancer. We recently discovered that during acquisition of resistance to HER2 inhibition, upregulation of the fatty acid transporter CD36 takes place, playing a key role in metabolic rewiring and resistance to anti-HER2 therapy.

Endocytosis of very low-density lipoproteins: an unexpected mechanism for lipid acquisition by breast cancer cells.

We previously described the expression of CD36 and LPL by breast cancer (BC) cells and tissues and the growth-promoting effect of VLDL observed only in the presence of LPL. We now report a model in which LPL is bound to a heparan sulfate proteoglycan motif on the BC cell surface and acts in concert with the VLDL receptor to internalize VLDLs via receptor-mediated endocytosis. We also demonstrate that gene-expression programs for lipid synthesis versus uptake respond robustly to triglyceride-rich lipoprotein availability. The literature emphasizes de novo FA synthesis and exogenous free FA uptake using CD36 as paramount mechanisms for lipid acquisition by cancer cells. We find that the uptake of intact lipoproteins is also an important mechanism for lipid acquisition and that the relative reliance on lipid synthesis versus uptake varies among BC cell lines and in response to VLDL availability. This metabolic plasticity has important implications for the development of therapies aimed at the lipid dependence of many types of cancer, in that the inhibition of FA synthesis may elicit compensatory upregulation of lipid uptake. Moreover, the mechanism that we have elucidated provides a direct connection between dietary fat and tumor biology.-.

CD36-Mediated Metabolic Rewiring of Breast Cancer Cells Promotes Resistance to HER2-Targeted Therapies.

Although it is established that fatty acid (FA) synthesis supports anabolic growth in cancer, the role of exogenous FA uptake remains elusive. Here we show that, during acquisition of resistance to HER2 inhibition, metabolic rewiring of breast cancer cells favors reliance on exogenous FA uptake over de novo FA synthesis. Through cDNA microarray analysis, we identify the FA transporter CD36 as a critical gene upregulated in cells with acquired resistance to the HER2 inhibitor lapatinib. Accordingly, resistant cells exhibit increased exogenous FA uptake and metabolic plasticity. Genetic or pharmacological inhibition of CD36 suppresses the growth of lapatinib-resistant but not lapatinib-sensitive cells in vitro and in vivo. Deletion of Cd36 in mammary tissues of MMTV-neu mice significantly attenuates tumorigenesis. In breast cancer patients, CD36 expression increases following anti-HER2 therapy, which correlates with a poor prognosis. Our results define CD36-mediated metabolic rewiring as an essential survival mechanism in HER2-positive breast cancer.