Unraveling ETC complex I function in ferroptosis reveals a potential ferroptosis-inducing therapeutic strategy for LKB1-deficient cancers.

The role of the mitochondrial electron transport chain (ETC) in regulating ferroptosis is not fully elucidated. Here, we reveal that pharmacological inhibition of the ETC complex I reduces ubiquinol levels while decreasing ATP levels and activating AMP-activated protein kinase (AMPK), the two effects known for their roles in promoting and suppressing ferroptosis, respectively. Consequently, the impact of complex I inhibitors on ferroptosis induced by glutathione peroxidase 4 (GPX4) inhibition is limited. The pharmacological inhibition of complex I in LKB1-AMPK-inactivated cells, or genetic ablation of complex I (which does not trigger apparent AMPK activation), abrogates the AMPK-mediated ferroptosis-suppressive effect and sensitizes cancer cells to GPX4-inactivation-induced ferroptosis. Furthermore, complex I inhibition synergizes with radiotherapy (RT) to selectively suppress the growth of LKB1-deficient tumors by inducing ferroptosis in mouse models. Our data demonstrate a multifaceted role of complex I in regulating ferroptosis and propose a ferroptosis-inducing therapeutic strategy for LKB1-deficient cancers.

3-Phosphoinositide-dependent kinase 1 drives acquired resistance to osimertinib.

Osimertinib sensitive and resistant NSCLC NCI-H1975 clones are used to model osimertinib acquired resistance in humanized and non-humanized mice and delineate potential resistance mechanisms. No new EGFR mutations or loss of the EGFR T790M mutation are found in resistant clones. Resistant tumors grown under continuous osimertinib pressure both in humanized and non-humanized mice show aggressive tumor regrowth which is significantly less sensitive to osimertinib as compared with parental tumors. 3-phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential driver of osimertinib acquired resistance, and its selective inhibition by BX795 and CRISPR gene knock out, sensitizes resistant clones. In-vivo inhibition of PDK1 enhances the osimertinib sensitivity against osimertinib resistant xenograft and a patient derived xenograft (PDX) tumors. PDK1 knock-out dysregulates PI3K/Akt/mTOR signaling, promotes cell cycle arrest at the G1 phase. Yes-associated protein (YAP) and active-YAP are upregulated in resistant tumors, and PDK1 knock-out inhibits nuclear translocation of YAP. Higher expression of PDK1 and an association between PDK1 and YAP are found in patients with progressive disease following osimertinib treatment. PDK1 is a central upstream regulator of two critical drug resistance pathways: PI3K/AKT/mTOR and YAP.

Patient-Derived Pancreatic Cancer Cells Induce C2C12 Myotube Atrophy by Releasing Hsp70 and Hsp90.

Pancreatic cancer (PC) patients are highly prone to cachexia, a lethal wasting syndrome featuring muscle wasting with an undefined etiology. Recent data indicate that certain murine cancer cells induce muscle wasting by releasing Hsp70 and Hsp90 through extracellular vesicles (EVs) to activate p38ß MAPK-mediated catabolic pathways primarily through Toll-like receptor 4 (TLR4). However, whether human PC induces cachexia through releasing Hsp70 and Hsp90 is undetermined. Here, we investigated whether patient-derived PC cells induce muscle cell atrophy directly through this mechanism. We compared cancer cells isolated from patient-derived xenografts (PDX) from three PC patients who had cachexia (PCC) with those of three early-stage lung cancer patients without cachexia (LCC) and two renal cancer patients who were not prone to cachexia (RCC). We observed small increases of Hsp70 and Hsp90 released by LCC and RCC in comparison to non-cancer control cells (NCC). However, PCC released markedly higher levels of Hsp70 and Hsp90 (~ 6-fold on average) than LCC and RCC. In addition, PCC released similarly increased levels of Hsp70/90-containing EVs. In contrast to RCC and LCC, PCC-conditioned media induced a potent catabolic response in C2C12 myotubes including the activation of p38 MAPK and transcription factor C/EBPß, upregulation of E3 ligases UBR2 and MAFbx, and increase of autophagy marker LC3-II, resulting in the loss of the myosin heavy chain (MHC ~50%) and myotube diameter (~60%). Importantly, the catabolic response was attenuated by Hsp70- and Hsp90-neutralizing antibodies in a dose-dependent manner. These data suggest that human PC cells release high levels of Hsp70 and Hsp90 that induce muscle atrophy through a direct action on muscle cells.

WNT5A-RHOA Signaling Is a Driver of Tumorigenesis and Represents a Therapeutically Actionable Vulnerability in Small Cell Lung Cancer.

WNT signaling represents an attractive target for cancer therapy due to its widespread oncogenic role. However, the molecular players involved in WNT signaling and the impact of their perturbation remain unknown for numerous recalcitrant cancers. Here, we characterize WNT pathway activity in small cell lung cancer (SCLC) and determine the functional role of WNT signaling using genetically engineered mouse models. ß-Catenin, a master mediator of canonical WNT signaling, was dispensable for SCLC development, and its transcriptional program was largely silenced during tumor development. Conversely, WNT5A, a ligand for ß-catenin-independent noncanonical WNT pathways, promoted neoplastic transformation and SCLC cell proliferation, whereas WNT5A deficiency inhibited SCLC development. Loss of p130 in SCLC cells induced expression of WNT5A, which selectively increased Rhoa transcription and activated RHOA protein to drive SCLC. Rhoa knockout suppressed SCLC development in vivo, and chemical perturbation of RHOA selectively inhibited SCLC cell proliferation. These findings suggest a novel requirement for the WNT5A-RHOA axis in SCLC, providing critical insights for the development of novel therapeutic strategies for this recalcitrant cancer. This study also sheds light on the heterogeneity of WNT signaling in cancer and the molecular determinants of its cell-type specificity. SIGNIFICANCE: The p130-WNT5A-RHOA pathway drives SCLC progression and is a potential target for the development of therapeutic interventions and biomarkers to improve patient treatment.

CombPDX: a unified statistical framework for evaluating drug synergism in patient-derived xenografts.

Anticancer combination therapy has been developed to increase efficacy by enhancing synergy. Patient-derived xenografts (PDXs) have emerged as reliable preclinical models to develop effective treatments in translational cancer research. However, most PDX combination study designs focus on single dose levels, and dose-response surface models are not appropriate for testing synergism. We propose a comprehensive statistical framework to assess joint action of drug combinations from PDX tumor growth curve data. We provide various metrics and robust statistical inference procedures that locally (at a fixed time) and globally (across time) access combination effects under classical drug interaction models. Integrating genomic and pharmacological profiles in non-small-cell lung cancer (NSCLC), we have shown the utilities of combPDX in discovering effective therapeutic combinations and relevant biological mechanisms. We provide an interactive web server, combPDX ( https://licaih.shinyapps.io/CombPDX/ ), to analyze PDX tumor growth curve data and perform power analyses.

Unbiased peptoid cell screen identifies a peptoid targeting newly appeared cell surface vimentin on tumor transformed early lung cancer cells.

To identify potential new reagents and biomarkers for early lung cancer detection we combined the use of a novel preclinical isogenic model of human lung epithelial cells comparing non-malignant cells with those transformed to full malignancy using defined oncogenic changes and our on-bead two color (red and green stained cells) (OBTC) peptoid combinatorial screening methodology. The preclinical model used normal parent lung epithelial cells (HBEC3-KT, labeled with green dye) and isogenic fully malignant transformed derivatives (labeled with a red dye) via the sequential introduction of key genetic alterations of p53 knockdown, oncogenic KRAS and overexpression of cMYC (HBEC3p53, KRAS, cMYC). Using the unbiased OBTC screening approach, we tested 100,000 different peptoids and identified only one (named JM3A) that bound to the surface of the HBEC3p53, KRAS, cMYC cells (red cells) but not HBEC3-KT cells (green cells). Using the JM3A peptoid and proteomics, we identified the protein bound as vimentin using multiple validation approaches. These all confirmed the cell surface expression of vimentin (CSV) on transformed (HBEC3p53, KRAS, cMYC) but not on untransformed (HBEC3-KT) cells. JM3A coupled with fluorophores was able to detect and stain cell surface vimentin on very early stage lung cancers but not normal lung epithelial cells in a fashion comparable to that using anti-vimentin antibodies. We conclude: using a combined isogenic preclinical model of lung cancer and two color screening of a large peptoid library, we have identified differential expression of cell surface vimentin (CSV) after malignant transformation of lung epithelial cells, and developed a new peptoid reagent (JM3A) for detection of CSV which works well in staining of early stage NSCLCs. This new, highly specific, easy to prepare, CSV detecting JM3A peptoid provides an important new reagent for identifying cancer cells in early stage tumors as well as a resource for detection and isolating of CSV expressing circulating tumor cells.

Combined MEK/MDM2 inhibition demonstrates antitumor efficacy in TP53 wild-type thyroid and colorectal cancers with MAPK alterations.

Most tumors with activating MAPK (mitogen-activated protein kinase) pathway alterations respond poorly to MEK inhibitors alone. Here, we evaluated combination therapy with MEK inhibitor selumetinib and MDM2 inhibitor KRT-232 in TP53 wild-type and MAPK altered colon and thyroid cancer models. In vitro, we showed synergy between selumetinib and KRT-232 on cell proliferation and colony formation assays. Immunoblotting confirmed p53 upregulation and MEK pathway inhibition. The combination was tested in vivo in seven patient-derived xenograft (PDX) models (five colorectal carcinoma and two papillary thyroid carcinoma models) with different KRAS, BRAF, and NRAS mutations. Combination therapy significantly prolonged event-free survival compared with monotherapy in six of seven models tested. Reverse-phase protein arrays and immunohistochemistry, respectively, demonstrated upregulation of the p53 pathway and in two models cleaved caspase 3 with combination therapy. In summary, combined inhibition of MEK and MDM2 upregulated p53 expression, inhibited MAPK signaling and demonstrated greater antitumor efficacy than single drug therapy in both in vitro and in vivo settings. These findings support further clinical testing of the MEK/MDM2 inhibitor combination in tumors of epithelial origin with MAPK pathway alterations.

Epstein Barr virus-positive B-cell lymphoma is highly vulnerable to MDM2 inhibitors in vivo.

Epstein-Barr virus-positive (EBV-positive) B-cell lymphomas are common in immunocompromised patients and remain an unmet medical need. Here we report that MDM2 inhibitors (MDM2is) navtemadlin and idasanutlin have potent in vivo activity in EBV-positive B-cell lymphoma established in immunocompromised mice. Tumor regression was observed in all 5 EBV-positive xenograft-associated B-cell lymphomas treated with navtemadlin or idasanutlin. Molecular characterization showed that treatment with MDM2is resulted in activation of p53 pathways and downregulation of cell cycle effectors in human lymphoma cell lines that were either EBV-positive or had undetectable expression of BCL6, a transcriptional inhibitor of the TP53 gene. Moreover, treatment with navtemadlin resulted in tumor regression and prevented systemic dissemination of EBV-positive lymphoma derived from 2 juvenile patients with posttransplant lymphoproliferative diseases, including 1 whose tumor was resistant to virus-specific T-cell therapy. These results provide proof-of-concept for targeted therapy of EBV-positive lymphoma with MDM2is and the feasibility of using EBV infection or loss of BCL6 expression to identify responders to MDM2is.

Structure-based classification predicts drug response in EGFR-mutant NSCLC.

Epidermal growth factor receptor (EGFR) mutations typically occur in exons 18-21 and are established driver mutations in non-small cell lung cancer (NSCLC)1-3. Targeted therapies are approved for patients with 'classical' mutations and a small number of other mutations4-6. However, effective therapies have not been identified for additional EGFR mutations. Furthermore, the frequency and effects of atypical EGFR mutations on drug sensitivity are unknown1,3,7-10. Here we characterize the mutational landscape in 16,715 patients with EGFR-mutant NSCLC, and establish the structure-function relationship of EGFR mutations on drug sensitivity. We found that EGFR mutations can be separated into four distinct subgroups on the basis of sensitivity and structural changes that retrospectively predict patient outcomes following treatment with EGFR inhibitors better than traditional exon-based groups. Together, these data delineate a structure-based approach for defining functional groups of EGFR mutations that can effectively guide treatment and clinical trial choices for patients with EGFR-mutant NSCLC and suggest that a structure-function-based approach may improve the prediction of drug sensitivity to targeted therapies in oncogenes with diverse mutations.

Comprehensive characterization of 536 patient-derived xenograft models prioritizes candidatesfor targeted treatment.

Development of candidate cancer treatments is a resource-intensive process, with the research community continuing to investigate options beyond static genomic characterization. Toward this goal, we have established the genomic landscapes of 536 patient-derived xenograft (PDX) models across 25 cancer types, together with mutation, copy number, fusion, transcriptomic profiles, and NCI-MATCH arms. Compared with human tumors, PDXs typically have higher purity and fit to investigate dynamic driver events and molecular properties via multiple time points from same case PDXs. Here, we report on dynamic genomic landscapes and pharmacogenomic associations, including associations between activating oncogenic events and drugs, correlations between whole-genome duplications and subclone events, and the potential PDX models for NCI-MATCH trials. Lastly, we provide a web portal having comprehensive pan-cancer PDX genomic profiles and source code to facilitate identification of more druggable events and further insights into PDXs' recapitulation of human tumors.

KEAP1 deficiency drives glucose dependency and sensitizes lung cancer cells and tumors to GLUT inhibition.

Metabolic reprogramming in cancer cells can create metabolic liabilities. KEAP1-mutant lung cancer is refractory to most current therapies. Here we show that KEAP1 deficiency promotes glucose dependency in lung cancer cells, and KEAP1-mutant/deficient lung cancer cells are more vulnerable to glucose deprivation than their WT counterparts. Mechanistically, KEAP1 inactivation in lung cancer cells induces constitutive activation of NRF2 transcription factor and aberrant expression of NRF2 target cystine transporter SLC7A11; under glucose limitation, high cystine uptake in KEAP1-inactivated lung cancer cells stimulates toxic intracellular disulfide buildup, NADPH depletion, and cell death, which can be rescued by genetic ablation of NRF2-SLC7A11 axis or treatments inhibiting disulfide accumulation. Finally, we show that KEAP1-inactivated lung cancer cells or xenograft tumors are sensitive to glucose transporter inhibitor. Together, our results reveal that KEAP1 deficiency induces glucose dependency in lung cancer cells and uncover a therapeutically relevant metabolic liability.

DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer.

Ferroptosis, a form of regulated cell death that is induced by excessive lipid peroxidation, is a key tumour suppression mechanism1-4. Glutathione peroxidase 4 (GPX4)5,6 and ferroptosis suppressor protein 1 (FSP1)7,8 constitute two major ferroptosis defence systems. Here we show that treatment of cancer cells with GPX4 inhibitors results in acute depletion of N-carbamoyl-L-aspartate, a pyrimidine biosynthesis intermediate, with concomitant accumulation of uridine. Supplementation with dihydroorotate or orotate-the substrate and product of dihydroorotate dehydrogenase (DHODH)-attenuates or potentiates ferroptosis induced by inhibition of GPX4, respectively, and these effects are particularly pronounced in cancer cells with low expression of GPX4 (GPX4low). Inactivation of DHODH induces extensive mitochondrial lipid peroxidation and ferroptosis in GPX4low cancer cells, and synergizes with ferroptosis inducers to induce these effects in GPX4high cancer cells. Mechanistically, DHODH operates in parallel to mitochondrial GPX4 (but independently of cytosolic GPX4 or FSP1) to inhibit ferroptosis in the mitochondrial inner membrane by reducing ubiquinone to ubiquinol (a radical-trapping antioxidant with anti-ferroptosis activity). The DHODH inhibitor brequinar selectively suppresses GPX4low tumour growth by inducing ferroptosis, whereas combined treatment with brequinar and sulfasalazine, an FDA-approved drug with ferroptosis-inducing activity, synergistically induces ferroptosis and suppresses GPX4high tumour growth. Our results identify a DHODH-mediated ferroptosis defence mechanism in mitochondria and suggest a therapeutic strategy of targeting ferroptosis in cancer treatment.

mTORC1 couples cyst(e)ine availability with GPX4 protein synthesis and ferroptosis regulation.

Glutathione peroxidase 4 (GPX4) utilizes glutathione (GSH) to detoxify lipid peroxidation and plays an essential role in inhibiting ferroptosis. As a selenoprotein, GPX4 protein synthesis is highly inefficient and energetically costly. How cells coordinate GPX4 synthesis with nutrient availability remains unclear. In this study, we perform integrated proteomic and functional analyses to reveal that SLC7A11-mediated cystine uptake promotes not only GSH synthesis, but also GPX4 protein synthesis. Mechanistically, we find that cyst(e)ine activates mechanistic/mammalian target of rapamycin complex 1 (mTORC1) and promotes GPX4 protein synthesis at least partly through the Rag-mTORC1-4EBP signaling axis. We show that pharmacologic inhibition of mTORC1 decreases GPX4 protein levels, sensitizes cancer cells to ferroptosis, and synergizes with ferroptosis inducers to suppress patient-derived xenograft tumor growth in vivo. Together, our results reveal a regulatory mechanism to coordinate GPX4 protein synthesis with cyst(e)ine availability and suggest using combinatorial therapy of mTORC1 inhibitors and ferroptosis inducers in cancer treatment.

PAF remodels the DREAM complex to bypass cell quiescence and promote lung tumorigenesis.

The DREAM complex orchestrates cell quiescence and the cell cycle. However, how the DREAM complex is deregulated in cancer remains elusive. Here, we report that PAF (PCLAF/KIAA0101) drives cell quiescence exit to promote lung tumorigenesis by remodeling the DREAM complex. PAF is highly expressed in lung adenocarcinoma (LUAD) and is associated with poor prognosis. Importantly, Paf knockout markedly suppressed LUAD development in mouse models. PAF depletion induced LUAD cell quiescence and growth arrest. PAF is required for the global expression of cell-cycle genes controlled by the repressive DREAM complex. Mechanistically, PAF inhibits DREAM complex formation by binding to RBBP4, a core DREAM subunit, leading to transactivation of DREAM target genes. Furthermore, pharmacological mimicking of PAF-depleted transcriptomes inhibited LUAD tumor growth. Our results unveil how the PAF-remodeled DREAM complex bypasses cell quiescence to promote lung tumorigenesis and suggest that the PAF-DREAM axis may be a therapeutic vulnerability in lung cancer.

Elevated NSD3 histone methylation activity drives squamous cell lung cancer.

Amplification of chromosomal region 8p11-12 is a common genetic alteration that has been implicated in the aetiology of lung squamous cell carcinoma (LUSC)1-3. The FGFR1 gene is the main candidate driver of tumorigenesis within this region4. However, clinical trials evaluating FGFR1 inhibition as a targeted therapy have been unsuccessful5. Here we identify the histone H3 lysine 36 (H3K36) methyltransferase NSD3, the gene for which is located in the 8p11-12 amplicon, as a key regulator of LUSC tumorigenesis. In contrast to other 8p11-12 candidate LUSC drivers, increased expression of NSD3 correlated strongly with its gene amplification. Ablation of NSD3, but not of FGFR1, attenuated tumour growth and extended survival in a mouse model of LUSC. We identify an LUSC-associated variant NSD3(T1232A) that shows increased catalytic activity for dimethylation of H3K36 (H3K36me2) in vitro and in vivo. Structural dynamic analyses revealed that the T1232A substitution elicited localized mobility changes throughout the catalytic domain of NSD3 to relieve auto-inhibition and to increase accessibility of the H3 substrate. Expression of NSD3(T1232A) in vivo accelerated tumorigenesis and decreased overall survival in mouse models of LUSC. Pathological generation of H3K36me2 by NSD3(T1232A) reprograms the chromatin landscape to promote oncogenic gene expression signatures. Furthermore, NSD3, in a manner dependent on its catalytic activity, promoted transformation in human tracheobronchial cells and growth of xenografted human LUSC cell lines with amplification of 8p11-12. Depletion of NSD3 in patient-derived xenografts from primary LUSCs containing NSD3 amplification or the NSD3(T1232A)-encoding variant attenuated neoplastic growth in mice. Finally, NSD3-regulated LUSC-derived xenografts were hypersensitive to bromodomain inhibition. Thus, our work identifies NSD3 as a principal 8p11-12 amplicon-associated oncogenic driver in LUSC, and suggests that NSD3-dependency renders LUSC therapeutically vulnerable to bromodomain inhibition.

Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts.

Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models. CNA inferences based on DNA sequencing and microarray data displayed substantially higher resolution and dynamic range than gene expression-based inferences, and they also showed strong CNA conservation from PTs through late-passage PDXs. CNA recurrence analysis of 130 colorectal and breast PT/PDX-early/PDX-late trios confirmed high-resolution CNA retention. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multiregion samples within patients. Our study demonstrates the lack of systematic copy number evolution driven by the PDX mouse host.