Increased spatial coupling of integrin and collagen IV in the immunoresistant clear cell renal cell carcinoma tumor microenvironment.

Background: Immunotherapy (IO) has improved survival for patients with advanced clear cell renal cell carcinoma (ccRCC), but resistance to therapy develops in most patients. We use cellular-resolution spatial transcriptomics in patients with IO naïve and IO exposed primary ccRCC tumors to better understand IO resistance. Spatial molecular imaging (SMI) was obtained for tumor and adjacent stroma samples. Spatial gene set enrichment analysis (GSEA) and autocorrelation (coupling with high expression) of ligand-receptor transcript pairs were assessed. Multiplex immunofluorescence (mIF) validation was used for significant autocorrelative findings and the cancer genome atlas (TCGA) and the clinical proteomic tumor analysis consortium (CPTAC) databases were queried to assess bulk RNA expression and proteomic correlates. Results: 21 patient samples underwent SMI. Viable tumors following IO harbored more stromal CD8+ T cells and neutrophils than IO naïve tumors. YES1 was significantly upregulated in IO exposed tumor cells. The epithelial-mesenchymal transition pathway was enriched on spatial GSEA and the associated transcript pair COL4A1-ITGAV had significantly higher autocorrelation in the stroma. Fibroblasts, tumor cells, and endothelium had the relative highest expression. More integrin αV+ cells were seen in IO exposed stroma on mIF validation. Compared to other cancers in TCGA, ccRCC tumors have the highest expression of both COL4A1 and ITGAV. In CPTAC, collagen IV protein was more abundant in advanced stages of disease. Conclusions: On spatial transcriptomics, COL4A1 and ITGAV were more autocorrelated in IO-exposed stroma compared to IO-naïve tumors, with high expression amongst fibroblasts, tumor cells, and endothelium. Integrin represents a potential therapeutic target in IO treated ccRCC.

Specifications of the ACMG/AMP guidelines for ACADVL variant interpretation.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is a relatively common inborn error of metabolism, but due to difficulty in accurately predicting affected status through newborn screening, molecular confirmation of the causative variants by sequencing of the ACADVL gene is necessary. Although the ACMG/AMP guidelines have helped standardize variant classification, ACADVL variant classification remains disparate due to a phenotype that can be nonspecific, the possibility of variants that produce late-onset disease, and relatively high carrier frequency, amongst other challenges. Therefore, an ACADVL-specific variant curation expert panel (VCEP) was created to facilitate the specification of the ACMG/AMP guidelines for VLCADD. We expect these guidelines to help streamline, increase concordance, and expedite the classification of ACADVL variants.

Therapy-associated remodeling of pancreatic cancer revealed by single-cell spatial transcriptomics and optimal transport analysis.

In combination with cell intrinsic properties, interactions in the tumor microenvironment modulate therapeutic response. We leveraged high-plex single-cell spatial transcriptomics to dissect the remodeling of multicellular neighborhoods and cell-cell interactions in human pancreatic cancer associated with specific malignant subtypes and neoadjuvant chemotherapy/radiotherapy. We developed Spatially Constrained Optimal Transport Interaction Analysis (SCOTIA), an optimal transport model with a cost function that includes both spatial distance and ligand-receptor gene expression. Our results uncovered a marked change in ligand-receptor interactions between cancer-associated fibroblasts and malignant cells in response to treatment, which was supported by orthogonal datasets, including an ex vivo tumoroid co-culture system. Overall, this study demonstrates that characterization of the tumor microenvironment using high-plex single-cell spatial transcriptomics allows for identification of molecular interactions that may play a role in the emergence of chemoresistance and establishes a translational spatial biology paradigm that can be broadly applied to other malignancies, diseases, and treatments.

Trackable Intratumor Microdosing and Spatial Profiling Provide Early Insights into Activity of Investigational Agents in the Intact Tumor Microenvironment.

PURPOSE: Cancer drug development is currently limited by a paradigm of preclinical evaluation that does not adequately recapitulate the complexity of the intact human tumor microenvironment (TME). To overcome this, we combined trackable intratumor microdosing (CIVO) with spatial biology readouts to directly assess drug effects in patient tumors in situ. EXPERIMENTAL DESIGN: In a first-of-its-kind phase 0 clinical trial, we explored the effects of an investigational stage SUMOylation-activating enzyme (SAE) inhibitor, subasumstat (TAK-981) in 12 patients with head and neck carcinoma (HNC). Patients scheduled for tumor resection received percutaneous intratumor injections of subasumstat and vehicle control 1 to 4 days before surgery, resulting in spatially localized and graded regions of drug exposure (∼1,000-2,000 µm in diameter). Drug-exposed (n = 214) and unexposed regions (n = 140) were compared by GeoMx Digital Spatial Profiler, with evaluation at single-cell resolution in a subset of these by CosMx Spatial Molecular Imager. RESULTS: Localized regions of subasumstat exposure revealed SUMO pathway inhibition, elevation of type I IFN response, and inhibition of cell cycle across all tumor samples. Single-cell analysis by CosMx demonstrated cell-cycle inhibition specific to the tumor epithelium, and IFN pathway induction commensurate with a TME shift from immune-suppressive to immune-permissive. CONCLUSIONS: Pairing CIVO with spatial profiling enabled detailed investigation of response to subasumstat across a diverse sampling of native and intact TME. We demonstrate that drug mechanism of action can be directly evaluated in a spatially precise manner in the most translationally relevant setting: an in situ human tumor.

Therapeutic resistance in acute myeloid leukemia cells is mediated by a novel ATM/mTOR pathway regulating oxidative phosphorylation.

While leukemic cells are susceptible to various therapeutic insults, residence in the bone marrow microenvironment typically confers protection from a wide range of drugs. Thus, understanding the unique molecular changes elicited by the marrow is of critical importance toward improving therapeutic outcomes. In this study, we demonstrate that aberrant activation of oxidative phosphorylation serves to induce therapeutic resistance in FLT3 mutant human AML cells challenged with FLT3 inhibitor drugs. Importantly, our findings show that AML cells are protected from apoptosis following FLT3 inhibition due to marrow-mediated activation of ATM, which in turn upregulates oxidative phosphorylation via mTOR signaling. mTOR is required for the bone marrow stroma-dependent maintenance of protein translation, with selective polysome enrichment of oxidative phosphorylation transcripts, despite FLT3 inhibition. To investigate the therapeutic significance of this finding, we tested the mTOR inhibitor everolimus in combination with the FLT3 inhibitor quizartinib in primary human AML xenograft models. While marrow resident AML cells were highly resistant to quizartinib alone, the addition of everolimus induced profound reduction in tumor burden and prevented relapse. Taken together, these data provide a novel mechanistic understanding of marrow-based therapeutic resistance and a promising strategy for improved treatment of FLT3 mutant AML patients.

High-plex imaging of RNA and proteins at subcellular resolution in fixed tissue by spatial molecular imaging.

Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .

Persistence of Adverse Drug Reaction-Related Hospitalization Risk Following Discharge.

This retrospective cohort study analyzed the administrative hospital records of 91,500 patients with the aim of assessing adverse drug reaction (ADR)-related hospital admission risk after discharge from ADR and non-ADR-related admission. Patients aged ≥18 years with an acute admission to public hospitals in Tasmania, Australia between 2011 and 2015 were followed until May 2017. The index admissions (n = 91,550) were stratified based on whether they were ADR-related (n = 2843, 3.1%) or non-ADR-related (n = 88,707, 96.9%). Survival analysis assessed the post-index ADR-related admission risk using (1) the full dataset, and (2) a matched subset of patients using a propensity score analysis. Logistic regression was used to identify the risk factors for ADR-related admissions within 90 days of post-index discharge. The patients with an ADR-related index admission were almost five times more likely to experience another ADR-related admission within 90 days (p < 0.001). An increased risk persisted for at least 5 years (p < 0.001), which was substantially longer than previously reported. From the matched subset of patients, the risk of ADR-related admission within 90 and 365 days more than doubled in the patients with an ADR-related index admission (p < 0.0001). These admissions were often attributed to the same drug class as the patients' index ADR-related admission. Cancer was a major risk factor for ADR-related re-hospitalization within 90 days; other factors included heart failure and increasing age.

Balloon assisted, ultrasound guided percutaneous thrombin injection of a large radial artery pseudoaneurysm using a trans-venous approach via an Ipsilateral Arteriovenous Fistula.

BACKGROUND: Pseudoaneurysm formation is known to complicate arteriovenous haemodialysis access. Ultrasound guided thrombin injection is a recognised treatment option, but is not possible in pseudoaneurysms with no measurable neck. Balloon assisted techniques have been described in such cases, which transiently obstruct flow out of the pseudoaneurysm and thereby prevent non-target embolization during ultrasound guided percutaneous thrombin injection. We describe a balloon assisted technique for the treatment of a radial artery pseudoaneurysm, via retrograde access from the draining cephalic vein of an arteriovenous fistula. METHOD: A 61-year-old male with a radio-cephalic fistula was found on duplex ultrasound to have a large radial artery pseudoaneurysm with no measurable neck, as well as a juxta-anastomotic cephalic vein stenosis. Endovascular treatment was selected over open surgery. Retrograde cephalic venous access was established, which allowed for concurrent treatment of both the venous stenosis and the arterial pseudoaneurysm. After balloon dilation of the juxta-anastomotic stenosis, a percutaneous transluminal angioplasty balloon catheter was advanced across the arteriovenous anastomosis and inflated across the neck of the radial artery pseudoaneurysm, to transiently obstruct blood flow. This allowed for safe injection of thrombin into the pseudoaneurysm by direct ultrasound guided sac puncture; thereby achieving thrombosis. CONCLUSIONS: Balloon assisted ultrasound guided thrombin injection is an endovascular treatment option that can obviate the need for open surgery in cases involving pseudoaneurysms with no measurable neck. The technique described allowed both concurrent treatment of a juxta-anastomotic venous stenosis and treatment of an arterial pseudoaneurysm from a single venous puncture. This technique avoided arterial access and its inherent complications.

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation.

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

Targeting Glutamine Metabolism and Redox State for Leukemia Therapy.

PURPOSE: Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the accumulation of immature myeloid precursor cells. AML is poorly responsive to conventional chemotherapy and a diagnosis of AML is usually fatal. More effective and less toxic forms of therapy are desperately needed. AML cells are known to be highly dependent on the amino acid glutamine for their survival. These studies were directed at determining the effects of glutaminase inhibition on metabolism in AML and identifying general weaknesses that can be exploited therapeutically. EXPERIMENTAL DESIGN: AML cancer cell lines, primary AML cells, and mouse models of AML and acute lymphoblastic leukemia (ALL) were utilized. RESULTS: We show that blocking glutamine metabolism through the use of a glutaminase inhibitor (CB-839) significantly impairs antioxidant glutathione production in multiple types of AML, resulting in accretion of mitochondrial reactive oxygen species (mitoROS) and apoptotic cell death. Moreover, glutaminase inhibition makes AML cells susceptible to adjuvant drugs that further perturb mitochondrial redox state, such as arsenic trioxide (ATO) and homoharringtonine (HHT). Indeed, the combination of ATO or HHT with CB-839 exacerbates mitoROS and apoptosis, and leads to more complete cell death in AML cell lines, primary AML patient samples, and in vivo using mouse models of AML. In addition, these redox-targeted combination therapies are effective in eradicating ALL cells in vitro and in vivo. CONCLUSIONS: Targeting glutamine metabolism in combination with drugs that perturb mitochondrial redox state represents an effective and potentially widely applicable therapeutic strategy for treating multiple types of leukemia.

Glutaminase inhibition improves FLT3 inhibitor therapy for acute myeloid leukemia.

Acute myeloid leukemia (AML) is a blood cancer that is poorly responsive to conventional cytotoxic chemotherapy and a diagnosis of AML is usually fatal. More effective and better-tolerated therapies for AML are desperately needed. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are one of the most frequently observed genetic defects in AML. FLT3 inhibitors have shown impressive anti-leukemic activity in clinical trials; however, sustained remissions using these inhibitors as monotherapy have not been achieved. Our previous studies have implicated impaired glutamine metabolism in response to FLT3 inhibitors as a dominant factor causing AML cell death. In this study, we have employed metabolic flux analysis to examine the effects of FLT3 inhibition on glutamine utilization in FLT3-mutated AML cells using stable isotope tracers. We found that the FLT3 inhibitor AC220 inhibited glutamine flux into the antioxidant factor glutathione profoundly due to defective glutamine import. We also found that the glutaminase inhibitor CB-839 similarly impaired glutathione production by effectively blocking flux of glutamine into glutamate. Moreover, the combination of AC220 with CB-839 synergized to deplete glutathione, induce mitochondrial reactive oxygen species, and cause loss of viability through apoptotic cell death. In vivo, glutaminase inhibition with CB-839 facilitated leukemic cell elimination by AC220 and improved survival significantly in a patient-derived xenograft AML mouse model. Therefore, targeting glutaminase in combination with FLT3 may represent an effective therapeutic strategy for improving treatment of FLT3-mutated AML.

Detection of membrane-bound and soluble antigens by magnetic levitation.

Magnetic levitation is a technique for measuring the density and the magnetic properties of objects suspended in a paramagnetic field. We describe a novel magnetic levitation-based method that can specifically detect cell membrane-bound and soluble antigens by measurable changes in levitation height that result from the formation of antibody-coated bead and antigen complex. We demonstrate our method's ability to sensitively detect an array of membrane-bound and soluble antigens found in blood, including T-cell antigen CD3, eosinophil antigen Siglec-8, red blood cell antigens CD35 and RhD, red blood cell-bound Epstein-Barr viral particles, and soluble IL-6, and validate the results by flow cytometry and immunofluorescence microscopy performed in parallel. Additionally, employing an inexpensive, single lens, manual focus, wifi-enabled camera, we extend the portability of our method for its potential use as a point-of-care diagnostic assay.

ATM/G6PD-driven redox metabolism promotes FLT3 inhibitor resistance in acute myeloid leukemia.

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML, they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism, resulting in impaired production of the antioxidant factor glutathione, which was further impaired by ATM or G6PD inactivation. Moreover, FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo, revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML.

Synthesis of an inositol hexakisphosphate (IP6) affinity probe to study the interactome from a colon cancer cell line.

Inositol hexakisphosphate (InsP6 or IP6) is an important signalling molecule in vesicular trafficking, neurotransmission, immune responses, regulation of protein kinases and phosphatases, activation of ion channels, antioxidant functions and anticancer activities. An IP6 probe was synthesised from myo-inositol via a derivatised analogue, which was immobilised through a terminal amino group onto Dynabeads. Systematic analysis of the IP6 interactome has been performed using the IP6 affinity probe using cytosolic extracts from the LIM1215 colonic carcinoma cell line. LC/MS/MS analysis identified 77 proteins or protein complexes that bind to IP6 specifically, including AP-2 complex proteins and ß-arrestins as well as a number of novel potential IP6 interacting proteins. Bioinformatic enrichment analysis of the IP6 interactome reinforced the concept that IP6 regulates a number of biological processes including cell cycle and division, signal transduction, intracellular protein transport, vesicle-mediated transport and RNA splicing.

Targeted single molecule mutation detection with massively parallel sequencing.

Next-generation sequencing (NGS) technologies have transformed genomic research and have the potential to revolutionize clinical medicine. However, the background error rates of sequencing instruments and limitations in targeted read coverage have precluded the detection of rare DNA sequence variants by NGS. Here we describe a method, termed CypherSeq, which combines double-stranded barcoding error correction and rolling circle amplification (RCA)-based target enrichment to vastly improve NGS-based rare variant detection. The CypherSeq methodology involves the ligation of sample DNA into circular vectors, which contain double-stranded barcodes for computational error correction and adapters for library preparation and sequencing. CypherSeq is capable of detecting rare mutations genome-wide as well as those within specific target genes via RCA-based enrichment. We demonstrate that CypherSeq is capable of correcting errors incurred during library preparation and sequencing to reproducibly detect mutations down to a frequency of 2.4 × 10(-7) per base pair, and report the frequency and spectra of spontaneous and ethyl methanesulfonate-induced mutations across the Saccharomyces cerevisiae genome.

Tyrosine kinase inhibition in leukemia induces an altered metabolic state sensitive to mitochondrial perturbations.

PURPOSE: Although tyrosine kinase inhibitors (TKI) can be effective therapies for leukemia, they fail to fully eliminate leukemic cells and achieve durable remissions for many patients with advanced BCR-ABL(+) leukemias or acute myelogenous leukemia (AML). Through a large-scale synthetic lethal RNAi screen, we identified pyruvate dehydrogenase, the limiting enzyme for pyruvate entry into the mitochondrial tricarboxylic acid cycle, as critical for the survival of chronic myelogenous leukemia (CML) cells upon BCR-ABL inhibition. Here, we examined the role of mitochondrial metabolism in the survival of Ph(+) leukemia and AML upon TK inhibition. EXPERIMENTAL DESIGN: Ph(+) cancer cell lines, AML cell lines, leukemia xenografts, cord blood, and patient samples were examined. RESULTS: We showed that the mitochondrial ATP-synthase inhibitor oligomycin-A greatly sensitized leukemia cells to TKI in vitro. Surprisingly, oligomycin-A sensitized leukemia cells to BCR-ABL inhibition at concentrations of 100- to 1,000-fold below those required for inhibition of respiration. Oligomycin-A treatment rapidly led to mitochondrial membrane depolarization and reduced ATP levels, and promoted superoxide production and leukemia cell apoptosis when combined with TKI. Importantly, oligomycin-A enhanced elimination of BCR-ABL(+) leukemia cells by TKI in a mouse model and in primary blast crisis CML samples. Moreover, oligomycin-A also greatly potentiated the elimination of FLT3-dependent AML cells when combined with an FLT3 TKI, both in vitro and in vivo. CONCLUSIONS: TKI therapy in leukemia cells creates a novel metabolic state that is highly sensitive to particular mitochondrial perturbations. Targeting mitochondrial metabolism as an adjuvant therapy could therefore improve therapeutic responses to TKI for patients with BCR-ABL(+) and FLT3(ITD) leukemias.

Inhibition of calcineurin combined with dasatinib has direct and indirect anti-leukemia effects against BCR-ABL1(+) leukemia.

Treatment of BCR-ABL1(+) leukemia has been revolutionized with the development of tyrosine kinase inhibitors. However, patients with BCR-ABL1(+) acute lymphoblastic leukemia and subsets of patients with chronic myeloid leukemia are at high risk of relapse despite kinase inhibition therapy, necessitating novel treatment strategies. We previously reported synthetic lethality in BCR-ABL1(+) leukemia cells by blocking both calcineurin/NFAT signaling and BCR-ABL1, independent of drug efflux inhibition by cyclosporine. Here, using RNA-interference we confirm that calcineurin inhibition sensitizes BCR-ABL1(+) cells to tyrosine kinase inhibition in vitro. However, when we performed pharmacokinetic and pharmacodynamic studies of dasatinib and cyclosporine in mice, we found that co-administration of cyclosporine increases peak concentrations and the area under the curve of dasatinib, which contributes to the enhanced disease control. We also report the clinical experience of two subjects in whom we observed more hematopoietic toxicity than expected while enrolled in a Phase Ib trial designed to assess the safety and tolerability of adding cyclosporine to dasatinib in humans. Thus, the anti-leukemia benefit of co-administration of cyclosporine and dasatinib is mechanistically pleiotropic, but may not be tolerable, at least as administered in this trial. These data highlight some of the challenges associated with combining targeted agents to treat leukemia.

Structural and mechanistic studies of polymerase η bypass of phenanthriplatin DNA damage.

Platinum drugs are a mainstay of anticancer chemotherapy. Nevertheless, tumors often display inherent or acquired resistance to platinum-based treatments, prompting the search for new compounds that do not exhibit cross-resistance with current therapies. Phenanthriplatin, cis-diamminephenanthridinechloroplatinum(II), is a potent monofunctional platinum complex that displays a spectrum of activity distinct from those of the clinically approved platinum drugs. Inhibition of RNA polymerases by phenanthriplatin lesions has been implicated in its mechanism of action. The present study evaluates the ability of phenanthriplatin lesions to inhibit DNA replication, a function disrupted by traditional platinum drugs. Phenanthriplatin lesions effectively inhibit DNA polymerases ν, ζ, and κ and the Klenow fragment. In contrast to results obtained with DNA damaged by cisplatin, all of these polymerases were capable of inserting a base opposite a phenanthriplatin lesion, but only Pol η, an enzyme efficient in translesion synthesis, was able to fully bypass the adduct, albeit with low efficiency. X-ray structural characterization of Pol η complexed with site-specifically platinated DNA at both the insertion and +1 extension steps reveals that phenanthriplatin on DNA interacts with and inhibits Pol η in a manner distinct from that of cisplatin-DNA adducts. Unlike cisplatin and oxaliplatin, the efficacies of which are influenced by Pol η expression, phenanthriplatin is highly toxic to both Pol η+ and Pol η- cells. Given that increased expression of Pol η is a known mechanism by which cells resist cisplatin treatment, phenanthriplatin may be valuable in the treatment of cancers that are, or can easily become, resistant to cisplatin.

Rational combination of a MEK inhibitor, selumetinib, and the Wnt/calcium pathway modulator, cyclosporin A, in preclinical models of colorectal cancer.

PURPOSE: The mitogen-activated protein kinase (MAPK) pathway is a crucial regulator of cell proliferation, survival, and resistance to apoptosis. MEK inhibitors are being explored as a treatment option for patients with KRAS-mutant colorectal cancer who are not candidates for EGFR-directed therapies. Initial clinical results of MEK inhibitors have yielded limited single-agent activity in colorectal cancer, indicating that rational combination strategies are needed. EXPERIMENTAL DESIGN: In this study, we conducted unbiased gene set enrichment analysis and synthetic lethality screens with selumetinib, which identified the noncanonical Wnt/Ca++ signaling pathway as a potential mediator of resistance to the MEK1/2 inhibitor selumetinib. To test this, we used shRNA constructs against relevant WNT receptors and ligands resulting in increased responsiveness to selumetinib in colorectal cancer cell lines. Further, we evaluated the rational combination of selumetinib and WNT pathway modulators and showed synergistic antiproliferative effects in in vitro and in vivo models of colorectal cancer. RESULTS: Importantly, this combination not only showed tumor growth inhibition but also tumor regression in the more clinically relevant patient-derived tumor explant (PDTX) models of colorectal cancer. In mechanistic studies, we observed a trend toward increased markers of apoptosis in response to the combination of MEK and WntCa(++) inhibitors, which may explain the observed synergistic antitumor effects. CONCLUSIONS: These results strengthen the hypothesis that targeting both the MEK and Wnt pathways may be a clinically effective rational combination strategy for patients with metastatic colorectal cancer.